Sour/Acid Taste Receptors Assays, Genes and Proteins

ABSTRACT

Taste receptor PC-1-L3/PC-2-L1 is provided. Methods and systems for screening for tastants and receptor modulators are provided. Knock out and transgenic animals, methods of detecting polymorphisms, and methods of correcting taste defects are also provided.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to and benefit of U.S. Ser. No.60/741,352, TASTE RECEPTOR GENES AND PROTEINS by Zuker and Huang, FiledNov. 30, 2005. This application is a CIP of and claims priority to U.S.Ser. No. 11/483,423 MAMMALIAN SOUR/ACID TASTE AND CSF RECEPTOR GENES,POLYPEPTIDES AND ASSAYS by Zuker and Huang filed Jul. 6, 2006. Each ofthese prior applications are incorporated herein by reference in theirentirety.

STATEMENT OF GOVERNMENT SUPPORT

This invention was partially supported by grant NIH R01 DC04861. TheUnited States government may have certain rights in the invention.

FIELD OF THE INVENTION

The invention includes the surprising discovery that PKD1-L3, previouslyimplicated as a potential polycystic kidney disease gene, encodes ataste receptor protein (denoted polycystin 1-like 3, or “PC-1-L3”).PC-1-L3 is a transmembrane ion channel protein that is co-expressed withtaste receptor protein polycystin 2-like 1, (“PC-2-L1”), encoded byPKD2-L1, in taste receptor cells.

BACKGROUND OF THE INVENTION

Taste transduction is one of the most sophisticated forms ofchemotransduction in animals (Avenet and Lindemann, 1989; Margolskee,1993; Lindemann, Physiol. Rev. 76:718-766, 1996; Kinnamon et al., Annu.Rev. Physiol. 54:715-731, 1992; and Gilbertson et al., Curr. Opin.Neurobiol. 10: 519-527, 2000). Gustatory signaling is found throughoutthe animal kingdom, from simple metazoans to the most complex ofvertebrates; its main purpose is to provide a reliable signalingresponse to non-volatile ligands.

Mammals are believed to have five basic types of taste modalities:salty, sour, sweet, umami (the taste of MSG), and bitter. Each of theseis thought to be mediated by distinct signaling pathways leading toreceptor cell depolarization, generation of a receptor or actionpotential and release of neurotransmitter and synaptic activity (Roper(1989) Ann. Rev. Neurosci. 12:329-353).

In general, the identification of new taste receptors is highlydesirable. The identification of a taste receptor provides methods andsystems for screening for new tastants, such as the identification ofnew artificial tastants (sweeteners, sour flavors, salt substitutes,etc.) and for the identification of activity modulators that produce agreater receptor response to specified amounts of a tastant. Forexample, the use of sour or other flavor enhancers may be useful inreducing the amount of sour or other flavoring needed to provoke,enhance, reduce or eliminate a sour receptor taste cell response, whichmay thus be useful as a flavor modulator. Similarly, acid is used as apreservative; the ability to reduce the flavor impact of suchpreservatives can be useful in food storage and packaging applications.

Relatively recently, the receptors for bitter, sweet and umami werecloned and shown to be encoded by two families of G-protein coupledreceptors (Nelson et al., 2000; Nelson et al., 2001; Zhang et al., 2003;Zhao et al., 2003; Mueller et al., 2005). In contrast, most of themolecular components of the sour pathways are previously unknown.Electrophysiological studies suggested that sour tastants modulate tastecell function by direct entry of H⁺ and Na⁺ ions through specializedmembrane channels on the apical surface of the cell. Thus, ion channelsselectively expressed in taste receptor cells could be candidates formediators of sour/acid tastes. Alternatively, ion channels can functionas a final critical signaling component in the activation of taste cells(akin to the role of TRPM5 in sweet, umami and bitter cells; Zhang etal., 2003).

Many other families of cell receptors are also known to function in avariety of signal transduction events associated with cell sensation.For example, the polycystins (e.g., polycystin-1, or “PC-1” andpolycystin-2, or “PC-2,” encoded by PKD1 and PKD2, respectively) areintegral membrane proteins with large extracellular N termini that arethought to possess a number of functions, including mechanosensation inrenal and nodal cilia (reviewed in Nauli and Zhou 2004 “Polycystins andMechanosensation in renal and nodal cilia” Bioessays 26.8 844-856 WileyPeriodicals). The polycystins fall into two basic classes of proteins,the PC-1-like proteins, which are receptor-like molecules and thePC-2-like proteins, which are ion channels (these proteins can alsocollectively form ion channel pore complexes). Several studies havefound overlapping and interdependent roles for these proteins in varioussystems, particularly in kidney cells. Mutations in various of thesegenes cause polycystic kidney disease.

The present invention includes the surprising discovery that certain ofthe polycystin genes encode taste receptor proteins.

SUMMARY OF THE INVENTION

The invention includes the surprising discovery that PKD1-L3, encodingpolycystin 1-L3 (PC-1-L3), is a sour/acid taste receptor protein.Applicants previously described the surprising discovery that PKD2-L1,which encodes polycystin 2-L1 (PC-2-L1), is a sour/acid taste receptorprotein. See, co-pending application U.S. Ser. No. 11/176,958 and U.S.Ser. No. 11/483,423, incorporated herein by reference for all purposes.PC-1-L3 has been found to be a likely partner for polycystin-2L1 insome, though not all tissues expressing PKD2L1, including certain tastereceptor cells. PKD1-L3 and PKD2-L1 are co-expressed in taste receptorcells in vivo and their products interact in sour/acid taste signalingin those cells. The surprising discovery that PC-1-L3 and PC-2-L1 arespecifically co-expressed in certain taste receptor cells, suggestingthat they form taste receptor protein complexes (e.g., including PC-1-L3and/or PC-2-L1) in those cells (e.g., in the form of receptors and/orion channels and/or receptor/channel complexes) provides receptortargets for tastant and activity modulator identification and forstudies on any taste-related physiological or behavioral effectsmediated by either of these polypeptides, separately, and/or incombination.

Previously, PC-1-L3 and PC-2-L1 were though to be primarily involved inkidney function, as defects in various closely related PKD1 and PKD2genes (encoding PC-1 and PC-2 proteins) are known to cause polycystickidney disease. The surprising discovery that PC-1-L3 and PC-2-L1 arespecifically co-expressed in taste receptor cells, suggesting that theyform taste receptor protein complexes (e.g., including PC-1-L3 and/orPC-2-L1) in those cells (e.g., in the form of receptors and/or ionchannels and/or receptor/channel complexes) provides receptor targetsfor tastant and activity modulator identification and for studies on anytaste-related behavioral effects mediated by these proteins, separately,and/or in combination. The PC-2-L1 receptor protein has beendefinitively assigned as the sour/acid receptor (see, e.g., U.S. Ser.No. 11/483,423), as well as having a role in CNS acid receptorsensation. Based on the association between PC-2-L1 and PC-1-L3 incertain taste receptors, PC-1-L3 is assigned as a component of thesour/acid sensation pathway.

Assays of the invention can be cell or tissue based, e.g., screening ofnatural or transgenic cells, or transfected cells or tissues expressingPKD1-L3, PKD2L1, PC-2-L1 and/or PC-1-L3 for activity in response to testcompounds, or can be behaviorally based on whole animal studies. Foranimal studies, transgenic non-human animals (e.g., mice) can beproduced, including PKD1-L3 and/or PKD2-L1 knock-outs and transgenicanimals comprising heterologous PKD1-L3 and/or PKD2-L1 genes, e.g., tofacilitate behavioral and tastant studies for PKD1-L3 and/or PKD2-L1gene(s) and encoded proteins of interest. For example, a PKD1-L3 and/orPKD2-L1 knock-out mouse can be made transgenic with the PKD2-L1 and/orPKD1-L3 gene from a human, and the resulting transgenic mouse used tostudy responses to putative human PC-2-L1 and/or PC-1-L3 binders andactivity modulators. In addition, the invention provides for theidentification of taste-receptor defects at the molecular level (e.g.,thorough detection of PKD1-L3 and/or PKD2-L1 polymorphisms) and for thecorrection of these defects by gene therapy. Corresponding systems andkits are also included. Further details regarding these and otherfeatures of the invention are found herein.

Thus, in a first aspect, the invention provides methods of identifying acompound that binds to and/or modulates an activity of a PC-1-L3receptor polypeptide, or a PC-2-L1/PC-1-L3 polypeptide complex.Typically, the method includes contacting a biological or biochemicalsample comprising the polypeptide or complex with a test compound.Binding of the test compound to the PC-1-L3 receptor polypeptide orPC-2-L1/PC-1-L3 polypeptide complex, and/or modulation of the activityof the polypeptide or polypeptide complex by the test compound isdetected. This identifies the compound that binds to and/or modulatesthe activity of the PC-1-L3 receptor polypeptide and/or complex.

In a closely related aspect, the invention provides methods of screeningfor a compound that binds to and/or modulates an activity of a PC-1-L3taste receptor polypeptide, or a PC-2-L1/PC-1-L3 polypeptide complex.The method includes contacting a biological or biochemical samplecomprising the PC-1-L3 taste receptor polypeptide or PC-2-L1/PC-1-L3polypeptide complex with a test compound, and determining whether thetest compound binds to the PC-1-L3 taste receptor polypeptide orPC-2-L1/PC-1-L3 polypeptide complex, and/or modulates an activity of thepolypeptide or polypeptide complex by the test compound, therebyscreening the compound for binding to and/or modulation of the PC-1-L3taste receptor polypeptide or PC-2-L1/PC-1-L3 polypeptide complex.

Related methods of modulating an activity of a cell expressing apolycystin-1L3 or polycystin 2L1-polycystin 1L3 polypeptide or complex,are also a feature of the invention. These methods include contactingthe cell with a compound that binds to or modulates an activity of thepolypeptide or complex, e.g., as identified herein. In addition tomodulating activity, such compounds can be used for labeling the cell todetect PC-2-L1, PC-1-L3, complexes thereof, PKD1L3 and/or PKD2L1, e.g.,using in situ hybridization experiments. Examples of modulators/labelsinclude antibodies against the polypeptide or complex and nucleic acidsthat hybridize to PKD1L3 or PKD2L1 (including probes, anti-sense RNAs,SiRNAs, RNAs, tncRNAs, smRNAs, and/or other probes or DNA or RNAinterference moieties).

For these methods, the biological sample can be moved into contact withthe test compound, or vice versa, depending on the format of the methodthat is selected. For example, either the test compound or thepolypeptide can be fixed in position, e.g., in a solid phase or liquidphase array, and the appropriate polypeptide or test compound can becontacted to the fixed component. Alternately, both polypeptide and testcompound can be in a mobile phase, e.g., in a microfluidic device. Thus,“contacting” in these methods refers to the polypeptide and testcompound being brought into contact with each other, regardless of whichcomponent is moved to achieve contact of the relevant components.

Typically, where the assay methods of the invention are cell-based, suchassays can be used in a high throughput fashion to screen one or usepreparations of cellular materials. In these embodiments, the biologicalsample comprises or is derived from a cell that expresses thepolycystin-2L1 and/or polycystin-1L3 taste receptor polypeptide(s). Mosttypically, such cells are provided by expressing an appropriate gene ina recombinant cell. The gene is typically, though not necessarily,heterologous to the recombinant cell. Binding of the test compounds tothe receptor polypeptide(s)/complex, or modulation of the activity ofthe polypeptide(s)/complex by the test compounds is detected, e.g., in ahigh throughput screen, thereby identifying compounds that bind to ormodulate the activity of polypeptide(s). The polypeptide(s) can includetaste receptor polypeptides, pH sensing receptor polypeptides (includingin the CSF, where PC-2-L1 is an acid sensor), and/or the like.

Regardless of assay format, one or more biological sample comprising oneor more PC-1-L3 receptor polypeptide or PC-2-L1/PC-1-L3 polypeptidecomplex can be contacted with a plurality of test compounds. Binding ofthe test compounds to the PC-1-L3 receptor polypeptide orPC-2-L1/PC-1-L3 polypeptide complex, or modulation of the activity ofthe polypeptide by the test compounds can then be detected, therebyidentifying one or more compound that binds to or modulates the activityof the PC-1-L3 taste receptor polypeptide or PC-2-L1/PC-1-L3 polypeptidecomplex. High throughput cell or in vitro formats that achieve testingof 100, 1,000, 10,000 or more test compounds per hour can be used, ascan lower throughput formats, such as in vivo assays using heterologousmice. Test compounds can be pre-screened for any desired property,including toxicity, biodistribution, oral availability, or the like.

The test compounds can be any of a variety of different compounds,including naturally occurring compounds, ions, sour tastants, smallorganic molecules, peptides, peptide mimetics, an acid, a weak acid, CO,CO₂, acetic acid, a specific blocker of carbonic anhydrase, MK-417, anion channel agonist, an ion channel antagonist, an ion channel enhancer,a non-specific Ca⁺ channel blocker, Nifedipine and structurally relatedcompounds, Verapamil and structurally related compounds, gadolinium andstructurally related compounds, a stretch-induced channel blocker, anantibody to the polypeptide or complex, or the like. In one preferredembodiment, the test compound enhances an activity of the PC-1-L3 tastereceptor polypeptide or PC-2-L1/PC-1-L3 polypeptide complex, e.g., bypotentiating an activity of the PC-1-L3 taste receptor polypeptide orPC-2-L1/PC-1-L3 polypeptide complex. In another preferred aspect, thetest compound inhibits or blocks an activity of the PC-1-L3 tastereceptor polypeptide or PC-2-L1/PC-1-L3 polypeptide complex.

The biological sample from which the PC-1-L3 and/or PC-2-L1 polypeptidesare derived can include or be derived from a cell that expresses thePC-1-L3 taste receptor polypeptide or the PC-2-L1/PC-1-L3 complex. Theprecise source of PC-1-L3 used in the methods and compositions hereinwill vary depending on the application. For example, a human polypeptidecan be used in in vitro or in vivo tastant and/or modulator studies todetermine the likely effects of test compounds in human populations.Thus, the PC-1-L3 taste receptor polypeptide is optionally anypolypeptide homologous to, e.g., the human or murine PC-1-L3, includingthe human and/or murine PC-1-L3 taste receptor polypeptide(s).Similarly, the PC-2-L1 in a PC-2-L1/PC-1-L3 complex can be any suchpolypeptide, resulting, e.g., in a human or murine PC-2-L1/PC-1-L3polypeptide complex (or a complex with heterologous components, such asa human/murine peptide complex). Polypeptides derived from humans,laboratory animals and/or domesticated or livestock animals are usefulin the various methods herein, as are those derived from wild animals.Here again, the selection of the most appropriate polypeptide depends onthe intended end-application for information of tastant or modulationactivity of a test compound of interest. For example, where modulatorsof the receptor in humans are sought, human proteins can be used, e.g.,in a cell-based system where the genes for the proteins arerecombinantly expressed in the cell, or, e.g., in a transgenic modelsystem suitable for tastant or modulator analysis, such as a knock-outmouse that expresses the human proteins. Similarly, in veterinaryapplications, recombinant or transgenic models can be used to assesstastants or modulators, e.g., for livestock applications.

In one aspect, the biological source of PC-1-L3 or PC-2-L1 can be aPKD1-L3 or a PKD2L1 gene expressed in a recombinant cell, or the PKD1-L3gene and PKD2L1 genes can both be expressed in a recombinant cell.Generally, the PKD-L3 gene, the PKD2-L1 gene or both the PKD1-L3 and thePKD2L1 gene is or are heterologous to the recombinant cell (e.g., ahuman, rodent or insect cell), which can be in culture, in vivo, derivedfrom a cultured or in vivo cell (e.g., a primary cell, such as a cellderived from a taste bud or kidney cell) or the like.

A variety of different tastant/modulator/moieties can be detected forbinding or activity on PC-1-L3 and/or PC-1-L3/PC-2-L1 complexes. Forexample, the methods can include detecting binding between the PC-1-L3taste receptor polypeptide and a moiety such as a PC-2-L1 polypeptide, apotentiator of the PC-1-L3 taste receptor polypeptide, an antagonist ofthe PC-1-L3 taste receptor polypeptide, an agonist of the PC-1-L3 tastereceptor polypeptide, an inverse agonist of the PC-1-L3 taste receptorpolypeptide, a ligand that specifically binds to the PC-1-L3 tastereceptor polypeptide, and/or an antibody that specifically binds to thePC-1-L3 taste receptor polypeptide. Similarly, the methods optionallyinclude detecting binding between the PC-2-L1/PC-1-L3 polypeptidecomplex and a moiety such as: a potentiator of the complex, anantagonist of the complex, an agonist of the complex, an inverse agonistof the complex, a ligand that specifically binds to the complex, and anantibody that specifically binds to the complex.

Binding or activity can be detected, e.g., in vitro, in situ or in vivo,and optionally includes detection of the PC-1-L3 taste receptorpolypeptide or the PC-2-L1/PC-1-L3 polypeptide complex. Alternately (oradditionally) a signal resulting from an activity of the PC-1-L3 tastereceptor polypeptide or the PC-2-L1/PC-1-L3 polypeptide complex can bedetected. The signal can be, e.g., a conformation-dependent signal,e.g., where a conformation of the PC-1-L3 taste receptor polypeptide orthe PC-2-L1/PC-1-L3 polypeptide complex is modified by binding of thetest compound to the PC-1-L3 taste receptor polypeptide or to thePC-2-L1/PC-1-L3 polypeptide complex.

Detecting binding of a test compound to the PC-1-L3 taste receptorpolypeptide or the PC-2-L1/PC-1-L3 polypeptide complex, or activity ofthe test compound on the PC-1-L3 taste receptor polypeptide or thePC-2-L1/PC-1-L3 polypeptide complex can be performed in a variety ofdifferent formats. These include detecting binding between PC-2-L1 andPC-1-L3, formation or stability of the polypeptide complex, H⁺ flux, Na⁺flux, Ca²⁺ flux, ion flux, changes in an activity of an intracellular pHor ion sensor, depolarization of the cell, cell membrane voltagechanges, cell membrane conductivity changes, a kinase activity triggeredupon binding of a compound to the PC-1-L3 taste receptor polypeptide,generation, breakdown or binding of a phorbol ester by the PC-1-L3 tastereceptor polypeptide, binding of diacylglycerol or other lipids by thePC-1-L3 taste receptor polypeptide, cAMP activity, cGMP activity,GTPgammaS binding, phospholipase C activity, activity of an enzymeinvolved in cellular ionic balance, binding of PC-1-L3 to another PKDprotein, and/or a transcriptional reporter activity.

In one aspect, the PC-1-L3 taste receptor polypeptide, the PC-2-L1polypeptide, or the PC-2-L1/PC-1-L3 complex is incorporated into abiosensor. Such a biosensor can incorporate standard sensing featuressuch as the use of a Chem-FET, readout, display, etc.

Systems for practicing these and other methods are also a feature of theinvention. For example, a system of the invention for detectingcompounds that bind to or modulate an activity of a PC-1-L3 tastereceptor polypeptide or PC-2-L1/PC-1-L3 complex is provided. The systemincludes, e.g., a biological sample comprising the PC-1-L3 tastereceptor polypeptide or the PC-2-L1/PC-1-L3 polypeptide complex, asource of a plurality of test compounds, and a detector capable ofdetecting binding of one or more of the test compounds to the PC-1-L3taste receptor polypeptide or PC-2-L1/PC-1-L3 polypeptide complex, ormodulation of the activity of the polypeptide or complex by one or moreof the test compounds. This provides for the identification of aputative tastant compound that binds to or modulates the activity of thepolypeptide or complex.

The source of biological samples, genes, test compounds, etc., can beany of those noted above with respect to methods of the invention. Inone aspect, the test compounds include a library of tastant compounds.This library can be of, e.g., compounds of interest, and can,optionally, be pre-screened or pre-selected for any desirable property(structure, binding to a PC protein, bioavailability, toxicity, etc.).

The detector can employ any available detection system, e.g., can be apatch clamp device, an optical detection device, or the like. Forexample, the detector can include a fluorescence detector that detectsFRET, changes in membrane potential or flow of a dye into or out of thecell.

In addition to, or in an embodiment of the methods and systems above,methods and systems for monitoring tastant-induced behavior in vivo,e.g., using an appropriate model system.

For example, the invention includes a method of detecting ataste-induced behavior modulated by a PC-1-L3 taste receptor polypeptideor PC-2-L1/PC-1-L3 taste receptor polypeptide complex. For example, themethods can include (a) introducing a heterologous PKD1-L3 tastereceptor gene into an animal such as a mouse and expressing an encodedheterologous PC-1-L3 taste receptor polypeptide in a taste bud of theanimal; (b) providing a putative taste receptor tastant or modulator tothe animal; and, (c) monitoring a feeding behavior of the animal inresponse to the presence of the putative taste receptor tastant.

A heterologous PKD2-L1 can be gene into the animal and an encodedpolycystin 2-L1 polypeptide in the taste bud, e.g., the resultingPC-2-L1/PC-1-L3 taste receptor polypeptide complex that forms in thetaste bud is heterologous to the animal. For example, the animal can bea mouse and the heterologous PKD1L3 and/or PKD2L1 gene can be a humanPKD1L3 taste receptor gene. The gene can include a heterologous promoterthat is active in the taste bud of the animal, such as a PKD2-L1promoter, a PKD1-L3 promoter, a T1R— gene promoter, T2R— gene promoter,TRPM5-gene promoter, a PLCB2 gene promoter, a repeater gene promoter, agustducin gene promoter, a Gi2 gene promoter, a cytokeratin-19 genepromoter, or a promoter for a gene that is naturally selectivelyexpressed in a taste receptor cell of the tongue or palate epithelium.

The precise format of the assay can vary, depending on availableinstrumentation. In one typical embodiment, a putative tastant ormodulator is provided on a licking device to the animal and lickingbehavior of the animal on the device is monitored, optionally inconjunction with the overall feeding behavior of the animal (e.g.,increases or decreases in feeding behaviors, e.g., feeding on sour oracidic foods, or the like).

Observations can be adjusted for various controls, or compared tobehavior changes induced by known tastants or modulators. For example,the putative tastant or modulator can be provided to the animal inconjunction with a control compound and the relative frequency offeeding behavior caused by the putative tastant can be compared to thecontrol compound.

Any of the various tastants or modulators noted above or herein can beapplied to these methods as well, e.g., the taste receptor tastant ormodulator can include an agonist, enhancer, antagonist, or inverseagonist of a PC-1-L3 polypeptide or the PC-2-L1/PC-1-L3 taste receptorpolypeptide complex.

Related systems for detecting a taste-induced behavior or physiologicaleffect modulated by a PC-1-L3 taste receptor polypeptide orPC-2-L1/PC-1-L3 taste receptor polypeptide complex are similarlyprovided. In one example, the system includes: (a.) a non-human animalcomprising a heterologous PKD1-L3 taste receptor gene that is expressedin a taste bud of the animal; (b.) a source of a putative tastant thatis accessible to the animal; and (c.) a detector that detects a feedingbehavior of the animal in response to the presence of the putativetastant.

Here again, the animal can be a knock-out mouse deficient in endogenousPC-1-L3 taste receptor polypeptide expression, e.g., a mouse thatexpresses a heterologous human PC-1-L3 taste receptor polypeptide.Optionally, the animal can be a double knockout deficient in endogenousPC-1-L3 taste receptor polypeptide expression and endogenous polycystin2-L1 expression, e.g., a mouse expressing a heterologous human PC-1-L3taste receptor polypeptide and a heterologous human polycystin 2-L1polypeptide.

The source can include, e.g., a lickable device, a fluid sourcecomprising the tastant, or a food source comprising the tastant. Thedetector can include, e.g., a camera that detects movement by theanimal. Optionally, the system further includes an analysis moduleoperably linked to the detector, e.g., an analysis module (e.g.,software in a computer) that analyzes information received from thedetector.

In another embodiment, the invention provides a recombinant cell thatincludes a heterologous PKD1-L3 gene and a heterologous PKD2-L1 gene. Asin other aspects herein, the cell can be, e.g., a human, rodent orinsect cell. Any of the various permutations of PKD genes noted hereinoptionally apply to this embodiment as well, e.g., wherein the PKD1-L3taste receptor gene or the heterologous PKD2-L1 gene are human, murine,or the like. Typically, the PKD1-L3 gene and the heterologous PKD2-L1are expressed in the cell and a polycystin 2-L1 polypeptide/PKD3-L1polypeptide complex is formed in the cell, or in or on a membrane of thecell.

Similarly, in a related aspect, the invention provides an isolated orrecombinant polypeptide complex that includes at least one of: arecombinant PC-1-L3 polypeptide and/or a recombinant polycystin 2-L1polypeptide. Typically, the complex further includes at least one of: aPC-1-L3 polypeptide, a polycystin 2-L1 polypeptide, a recombinantPC-1-L3 polypeptide and/or a recombinant polycystin 2-L1 polypeptide.For example, in one embodiment, an isolated or recombinant polypeptidecomplex of the invention includes recombinant human PC-1-L3 polypeptideand a recombinant human polycystin 2-L1 polypeptide. The isolated orrecombinant polypeptide can be (and typically is) expressed in one ormore recombinant cell(s).

Recombinant taste bud cells are also a feature of the invention. Forexample, a taste bud cell that includes a heterologous PKD1-L3 tastereceptor gene, or a heterologous PC-1-L3 taste receptor polypeptide is afeature of the invention. Optionally, the recombinant taste bud cellfurther includes a heterologous PKD2-L1 gene. The taste bud cell can be,e.g., present in a recombinant (e.g., transgenic) non-human animal.

A knock out non-human animal comprising a defect in a native PKD1-L3taste receptor gene or a defect in native PKD1-L3 gene expression isalso a feature of the invention. For example, a double knock-out animal,such as a mouse, deficient in endogenous PC-1-L3 taste receptorpolypeptide expression and endogenous polycystin 2-L1 expression isprovided. This knock-out animal can include a heterologous PKD1-L3 tastereceptor gene (and, optionally, a recombinant PKD2-L1 gene) that isexpressed in the tastebud of the animal. For example, a PKD1-L3 and/orPKD2-L1 knock out mouse expressing a heterologous human PKD1-L3 geneand/or a PKD2-L1 gene is a feature of the invention.

Methods of detecting a molecular basis for a taste receptor functionabnormality are also provided. The methods include determining whether abiological sample from a patient comprises a polymorphism in a geneencoding PKD1-L3 or an abnormality in expression of PKD1-L3. Thepolymorphism is correlated with an abnormality in taste receptorfunction, thereby determining whether the patient has a genetic basisfor a taste receptor function abnormality. For example, the polymorphismcan be a single nucleotide polymorphism, a rearrangement, a splicingvariant, an expression variant, or the like. For example, theabnormality can be a variation in the expression of PKD1-L3 such as anabnormal tissue distribution of PKD1-L3 mRNA or PC-1-L3 polypeptide inthe organism at issue.

Methods of rescuing a taste receptor cell that has altered or missingPKD1-L3 taste receptor function, are provided. In these methods, anucleic acid encoding a recombinant polypeptide homologous to PC-1-L3(e.g., a naturally occurring PC-1-L3 gene) is introduced into the celland expressed. This provides PKD1-L3 function to the cell. Any of thevarious features noted herein can be applied to this embodiment as well,e.g., the cell can be in cell culture, in a tissue, in a taste bud, in amammal, etc.

Kits for practicing the above methods are also a feature of theinvention. The kits can include, e.g., a PKD1L3 and/or PKD2L1 nucleicacid, e.g., one or more vectors comprising a PKD1L3 and/or PKD2L1 gene,a PC-1-L3 and/or PC-2-L1 polypeptide, recombinant cells expressing thesegene or polypeptides, transgenic animals, etc., as noted above. The kitscan further include instructions for using the other kit components topractice the methods herein, system components, packaging materials forpackaging the components noted above, etc.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows results from the RNA in situ hybridization in circumvallatetaste papillae.

FIG. 2A-2B provide an alignments of sequences for human, rat, and mousePKD2L1.

FIG. 3 is photograph showing results demonstrating that PKD1L3selectively labels taste receptor cells.

FIG. 4 is an alignment of PKD1L3 sequences.

FIG. 5, Panels A show a series of traces showing the taste impact ofeliminating cells expressing PKD2L1 in the tongue. Note the total lossof sour taste in nerve responses. As a control also included are wildtype mice (upper traces) and engineered animals where sweet cells havebeen ablated (middle traces). Panel B shows histograms of response forwild-type and ablated animals.

FIG. 6, panels A-G shows PKD2L1 expression in cells surrounding thecentral canal.

FIG. 7A-C shows a diagram of the central canal, a labeledphotomicrograph and a series of traces showing pH responses ofPKD2L1-expressing cells surrounding the central canal.

FIG. 8, panels A and B show in situ hybridization images.

FIG. 9 shows antibody in situ hybridization results for binding ofantibodies to PKD1L3 and PKD2L1.

FIG. 10 illustrates loss of selective TRCs in DTA-expressing animals.Panel A Upper diagram illustrates the strategy used to target DTA or GFPto selective populations of TRCs. Panel B lower panels show in situhybridization experiments examining the presence of sweet (T1Rs), bitter(T2Rs) or PKD2L1-expressing cells in the two engineered lines.

DEFINITIONS

Before describing the present invention in detail, it is to beunderstood that this invention is not limited to particular technicalsystems, or biological components, which can, of course, vary. It isalso to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto be limiting. As used in this specification and the appended claims,the singular forms “a”, “an” and “the” include plural referents unlessthe content clearly dictates otherwise.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the invention pertains. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice for testing of the present invention, the preferredmaterials and methods are described herein. In describing and claimingthe present invention, the following terminology will be used inaccordance with the definitions set out below.

A “polycystin-1L3 polypeptide” or “polycystin-1L3 receptor polypeptide”is a polypeptide that is the same as, a splice-variant of, or homologousto a human polycystin-1L3 or murine polycystin-1L3, or that is derivedfrom such a polypeptide (e.g., through cloning, recombination, mutation,or the like). The polypeptide can be full length or can be a fragment ofa full length protein. A polycystin-1L3 fragment typically includes atleast 10 contiguous amino acids corresponding to a native polycystin-1L3protein, such as a human murine, dog or rat polycystin-1L3. Thepolycystin-1L3 receptor polypeptide can be naturally occurring orrecombinant, and can be unpurified, purified, or isolated, and canexist, e.g., in vitro, in vivo, or in situ. In one typical usefulembodiment, the polycystin-1L3 receptor polypeptide is a transmembraneprotein. As described herein, in useful embodiments, the polycystin-1L3receptor polypeptide can be a component of a sour/acid receptor, in anyof a variety of contexts, including as a taste receptor polypeptide, asa CSF receptor polypeptide or as an acid receptor in other systems,e.g., in the kidney.

A “polycystin-1L3 taste receptor polypeptide” is a polypeptide that isthe same as, a splice-variant of, or homologous to a humanpolycystin-1L3 or murine polycystin-1L3 and that is expressed in tastereceptor cells, or that is derived from such a polypeptide that isexpressed in such taste receptor cells (e.g., through cloning,recombination, mutation, or the like). The polypeptide can be fulllength or can be a fragment of a full length protein. A polycystin-1L3fragment typically includes at least 10 contiguous amino acidscorresponding to a native polycystin-1L3 protein, such as a humanmurine, dog or rat polycystin-1L3. The polycystin-1L3 taste receptorpolypeptide can be naturally occurring or recombinant, and can beunpurified, purified, or isolated, and can exist, e.g., in vitro, invivo, or in situ. In one typical useful embodiment, the polycystin-2L1taste receptor polypeptide is a transmembrane protein.

A “polycystin-2L1 polypeptide” or a polycystin-2L1 receptor polypeptideis a polypeptide that is the same as, a splice-variant of, or homologousto a human polycystin-2L1 or murine polycystin-2L1, or that is derivedfrom such a polypeptide (e.g., through cloning, recombination, mutation,or the like). The polypeptide can be full length or can be a fragment ofa full length protein. A polycystin-2L1 fragment typically includes atleast 10 contiguous amino acids corresponding to a native polycystin-2L1protein, such as a human murine, dog or rat polycystin-2L1. Thepolycystin-2L1 receptor polypeptide can be naturally occurring orrecombinant, and can be unpurified, purified, or isolated, and canexist, e.g., in vitro, in vivo, or in situ. In one typical usefulembodiment, the polycystin-2L1 receptor polypeptide is a transmembraneprotein. As described herein, in useful embodiments, the polycystin-2L1receptor polypeptide can be a sour/acid receptor, in any of a variety ofcontexts, including as a taste receptor polypeptide, as a CSF receptorpolypeptide or as an acid receptor in other systems, e.g., in thekidney.

A “polycystin-2L1 taste receptor polypeptide” is a polypeptide that isthe same as, a splice-variant of, or homologous to a humanpolycystin-2L1 or murine polycystin-2L1 and that is expressed in tastereceptor cells, or that is derived from such a polypeptide that isexpressed in such taste receptor cells (e.g., through cloning,recombination, mutation, or the like). The polypeptide can be fulllength or can be a fragment of a full length protein. A polycystin-2L1fragment typically includes at least 10 contiguous amino acidscorresponding to a native polycystin-2L1 protein, such as a humanmurine, dog or rat polycystin-2L1. The polycystin-2L1 taste receptorpolypeptide can be naturally occurring or recombinant, and can beunpurified, purified, or isolated, and can exist, e.g., in vitro, invivo, or in situ. In one typical useful embodiment, the polycystin-2L1taste receptor polypeptide is a transmembrane protein.

A “PKD1L3 gene” is a nucleic acid that encodes a PC-1-L3 polypeptide.Typically, the gene includes regulatory sequences that direct expressionof the gene in one or more cells of interest. Optionally, the PKD1L3gene is a native gene that includes regulatory and coding sequences thatnaturally direct expression of a PC-1-L3 polypeptide.

A “PKD2L1 gene” is a nucleic acid that encodes a PC-2-L1 polypeptide.Typically, the gene includes regulatory sequences that direct expressionof the gene in one or more cells of interest. Optionally, the PKD2L1gene is a native gene that includes regulatory and coding sequences thatnaturally direct expression of a PC-2-L1 polypeptide.

A biological sample comprising the PC-2-L1 and/or PC-1-L3 polypeptideincludes any sample comprising the PC-2-L1 and/or PC-1-L3 polypeptidethat is derived from a biological source, e.g., cells, tissues,organisms, cells, secretions, etc. These samples can include, e.g.,cells expressing the receptor, membranes containing the receptor,receptor bound to a chemical matrix, receptor bound to solid surface(e.g., for plasmon resonance), etc. A biochemical source can includebiological sources and/or non-biological sources, such as purelysynthetic preparations of materials.

A “tastant” is a compound that can be tasted by the relevant organism.These typically include compounds that can stimulate or inhibit one ormore activity of one or more taste receptor, taste cells or othersensory cells and/or nerves in the oral cavity. A tastant can be anymolecule, including ions, peptides, nucleotides, natural compounds,small organic molecules, etc. that leads to modulation of taste receptoror taste cell activity or a change taste cell function either on its ownor in the presence of other compounds.

A “modulator” is a compound that modulates an activity of a givenpolypeptide, polypeptide complex or receptor, e.g., a taste receptorpolypeptide complex, e.g., in response to a tastant, or polypeptide orpolypeptide complex in response to a change in pH. The term “modulate”with respect to a polycystin-2L1 polycystin-1L3 polypeptide refers to achange in an activity or property of the polypeptide. For example,modulation can cause an increase or a decrease in a protein activity, abinding characteristic, membrane permeability or any other biological,functional, or immunological properties of such proteins. The change inactivity can arise from, for example, an increase or decrease inexpression of one or more genes that encode these proteins, thestability of an mRNA that encodes the protein, translation efficiency,or from a change in activity of the protein itself. For example, amolecule that binds to polycystin-2L1 or polycystin-1L3 can cause anincrease or decrease in a biological activity of the polypeptide.Example modulators include polycystin-2L1 or polycystin-1L3 allostericenhancers, agonists, antagonists, inverse agonists, or partial agonists,polycystin-2L1 or polycystin-1L3 ligands, antibodies to polycystin-2L1,polycystin-1L3, or complexes thereof, etc.

A taste receptor “modulator” is a compound that modulates an activity ofa taste receptor, e.g., in response to a tastant. The term “modulate”with respect to a PC-2-L1 and/or PC-1-L3 polypeptide refers to a changein an activity or property of the polypeptide or polypeptide complex.For example, modulation can cause an increase or a decrease in a proteinactivity, a binding characteristic, membrane permeability or any otherbiological, functional, or immunological properties of such proteins.The change in activity can arise from, for example, an increase ordecrease in expression of one or more genes that encode these proteins,the stability of an mRNA that encodes the protein, translationefficiency, or from a change in activity of the protein itself. Forexample, a molecule that binds to PC-1-L3 and/or PC-2-L1 can cause anincrease or decrease in a biological activity of the polypeptide orcomplex. Example modulators include PC-1-L3 and/or PC-2-L1 allostericenhancers, agonists, antagonists, inverse agonists, or partial agonists,PC-1L3 and/or PC-2-L1 ligands, antibodies to PC-1-L3, PC-2-L1, or acomplex of PC-1-L3 and PC-2-L1, etc.

A “prescreened” compound is a compound that is pre-selected for aproperty of interest, such as toxicity, lack of toxicity,bioavaliability, chemical structure, type of molecule (natural product,ion, ion channel agonist/antagonist/inverse agonist, etc.), or the like.For example, an “ingestible compound” is a compound that can be safelyingested in an amount that triggers a taste receptor or taste cellresponse by the compound. Certain compounds such as agonists orenhancers can have such a desired response when present at very lowdoses, while others are present in higher amounts. In addition, certainingestible compounds such as enhancers optionally have no taste of theirown, but enhance the action of natural or chemically synthesizedtastants on a taste receptor or taste cell.

A “transmembrane potential” is the work needed to move a unit of chargeacross a membrane such as a cell membrane.

A “cationic membrane permeable dye” is a dye which has a positive chargeunder specified pH (e.g., physiological pH) where the dye can cross aselected membrane such as the membrane of an intact cell.

An “anionic membrane permeable dye” is a dye which has a negative chargeat a specified pH (e.g., physiological pH) and which is membranepermeable and whose distribution between the inside and outside of thespace bounded by the membrane or between the inside and outside of themembrane, depends on the transmembrane potential across the membrane.

A “neutral dye” has an overall neutral charge under the relevantconditions at issue, e.g., a specified pH (e.g., physiological pH).

A “voltage sensing composition” is a transmembrane potential indicator,e.g., comprising a fluorescent dye. Common voltage sensing compositionsof the invention include one or more cationic or anionic membranepermeable dye(s).

A membrane is “depolarized” when the transmembrane potential across themembrane becomes more positive inside. A membrane is “hyperpolarized”when the transmembrane potential becomes more negative inside.

A membrane is “permeable” to a given component (dye, ion, etc.) whenthat component can cross the membrane. Permeability can be dependentupon the relevant conditions, e.g., temperature, ionic conditions,voltage potentials, or the like.

DETAILED DESCRIPTION

The ability to screen taste (and other) receptors for response tomodulators, tastants and taste receptor modulators is of considerablecommercial value. Libraries of putative tastant and/or modulatorcompounds can be screened for activity against a given receptor toidentify taste modulators, taste enhancers, sour tastants, and the like.For example, the identification of new sour/acid or other tastants is ofvalue, e.g., to provide new flavors that can be added to foods anddrinks. Similarly, compounds that modulate activity of a receptor can beused to make the receptor more (or less) sensitive to a tastant, whichis particularly valuable when considering responsiveness to sour/acid orother tastants that have associated health consequences uponconsumption. For example, many preservatives utilize acids, which impactflavors. The ability to neutralize flavor effects facilitates the use offood preservatives. In one aspect of the invention, PC-1-L3, PC-2-L1 orPC1-L3/PC2-L1 complexes are tested for responsiveness to sour or acidiccompounds, and modulators of the receptor are identified. Modulators ofthe genes for these proteins can similarly be tested for effects on theexpression of the proteins.

Given the identification of polycystin-2L1 as a sour/acid taste receptorprotein, and of polycystin-1L3 as a potential partner forpolycystin-2L1, there are several ways in which these proteins can nowbe screened for responsiveness to test compounds (tastants, activitymodulators, etc.). These include high-throughput cell-based assays,animal behavioral models (e.g., using transgenic animals that express ahuman or other desirable heterologous PC-1-L3, PC-2-L1 or PC1-L3/PC2-L1polypeptides), or the like. Modulators of the genes for thesepolypeptides can similarly be tested for effects on the expression ofthe proteins. Polymorphisms in the gene for PC-1-L3 (PKD1L3) and PC-2-L1(PKD2L1) can also be detected to provide a molecular test for tastingdisorders and defects in PKD1L3 and/or PKD2L1, which can also be rescuedby gene therapy. In this regard, administration of a gene therapy vectorto the tastebud is relatively simple, due to ready access to thistissue, and can be targeted to a considerable degree simply bycontrolling the site of vector administration. Systems and kits forpracticing the methods, transgenic animals (PKD1L3/PKD2L1 knock-outsand/or transgenics), and related features are also included within thescope of the invention. Further details regarding these various featuresof the invention are found herein.

In addition to the identification of PC-2L1 as a sour/acid tastereceptor, PC-2L1 has also been identified as a pH (e.g., acid level)sensor in certain neuronal cells that are in contact with the CSF (e.g.,part of the system that monitors blood CO₂). While PC-1-L3 has, thusfar, not been found in association with PC-2L1 in the CSF, it ispossible that PC-1-L3 may form part of an acid sensor in cells outsideof the taste system, e.g., in the CSF, kidney, or the like. Thisprovides, e.g., a basis for assays that screen for modulators of PC-1L3and complexes of PC-1-L3 with PC-2-L1 in the context of its role as anacid (pH) sensor outside of any function in sour/acid taste sensation.pH sensation regulates, e.g., respiration. Defects in respiration leadto a variety of disorders, including sudden infant death syndrome(SIDS), sleep apnea, persistent hiccups, fatigue, altitude sickness,hyperventilation, and many others. In addition, patients suffering fromtrauma, anesthesia, or surgery often develop difficulty breathing, whichmay benefit from administration of appropriate modulators.

Screening Test Compounds for Activity Against PC-1-L3. PC-2-L1 and/orPC-1-L3/PC-2L1

In one aspect, methods of identifying a compound that binds to ormodulates an activity of a PC-1-L3 or PC-2-L1 polypeptide, or aPC-1-L3/PC-2L1 polypeptide complex are provided. In these methods, abiological or biochemical sample comprising the polypeptide or complexis contacted with a test compound and binding of the test compound tothe polypeptide or complex, or modulation of the activity of thepolypeptide or complex by the test compound is detected, therebyidentifying the compound which binds to or modulates activity.

Desirably, a test compound can be, e.g., a potentiator or enhancer ofthe polypeptide and/or complex, an antagonist of the polypeptide and/orcomplex, an agonist of the polypeptide and/or complex, an inverseagonist of the polypeptide and/or complex, a ligand that specificallybinds to the polypeptide and/or complex, an antibody that specificallybinds to the polypeptide and/or complex, and/or the like.

Additional Details Regarding Screening Methods

High throughput methods of screening are particularly useful inidentifying tastants or modulators of activity, and/or of PKD1-L3 and/orPKD2L1 gene expression. Generally in these methods, one or morebiological sample that includes one or more PC-1-L3 and/or PC-2-L1 tastereceptor polypeptide(s) and/or genes is contacted with a plurality oftest compounds. Binding to PC-1-L3, PC-2-L1 or a complex of both PC-1-L3and PC-2-L1 or modulation of these polypeptides/complexes or genes bythe test compounds is detected, thereby identifying one or more compoundthat binds to or modulates activity of the peptide, complex and/or gene.

Essentially any available compound library can be screened in such ahigh-throughput format against a biological or biochemical sample, suchas a cell expressing a PC-1-L3 and/or PC-2-L1 polypeptide, and activityof the library members against the polypeptide(s) or expression thereofcan be assessed in a high-throughput fashion.

Many libraries of compounds are commercially available, e.g., from theSigma Chemical Company (Saint Louis, Mo.), Aldrich chemical company (St.Louis Mo.), and many can be custom synthesized by a wide range ofbiotech and chemical companies. A variety of proprietary libraries alsoexist, including those specifically designed for screening of tastereceptors, e.g., from Senomyx, Inc. (La Jolla Calif.).

In one desirable aspect, the plurality of test compounds comprise aplurality of compounds. Thus, the library to be screened can include apreviously unscreened library of compounds, or can include apre-screened library of compounds that are pre-screened for any propertythat is desired, e.g., toxicity, bioavialability, chemical structure,known activity (e.g., ion channel binding or modulating activity)ingestibility, or the like. Further details on available libraries arefound below.

In general, test compounds that enhance or potentiate an activity of therelevant polypeptide or complex are can be desirable, e.g., to enhancepH sensitivity for certain cells, e.g., to increase respiration, e.g.,to reduce altitude effects, or as flavor enhancers, and can be screenedfor using the methods of the invention. However, test compounds thatinhibits or block an activity of the polypeptide or complex are alsodesirable, e.g., where the taste sensation associated with a flavorwould benefit from reduced responsiveness (e.g., in those cases wheremore than usual of the tastant is desirably consumed), or in cases wheredecreased pH responsiveness are desirable (e.g., to reducehyperventilation). For example, reduced sensitivity to acid/sour tastescan be desirable, e.g., to permit greater use of acidic foodpreservatives.

Additional Details Regarding Assay Formats

In another aspect, the present invention relates to the use of PC-1-L3,PC-2-L1, complexes thereof and/or coding nucleic acids in methods foridentifying a compound, e.g., a tastant or modulator, thatinteracts/binds to the polypeptide or complex. The test compound(s) canbe any natural or synthetic molecule(s) such as ions, proteins orfragments thereof, carbohydrates, organic or inorganic compounds and/orthe like. For example, the test compounds can be naturally occurringcompounds, ions, sour tastants, small organic molecules, peptides,peptide mimetics, an acid, a weak acid, CO, CO₂, acetic acid, a specificblocker of carbonic anhydrase, MK-417, an ion channel agonist, an ionchannel antagonist, an ion channel enhancer, a non-specific Ca⁺ channelblocker, Nifedipine and structurally related compounds, Verapamil andstructurally related compounds, gadolinium and structurally relatedcompounds, a stretch-induced channel blocker, etc. This can be achieved,e.g., by utilizing the polypeptides of the invention, including activefragments thereof, in cell-free or cell-based assays. A variety offormats are applicable, including measurement of second messengereffects (e.g., H⁺ flux, Na⁺ flux, Ca²⁺ flux, ion flux, depolarization ofthe cell, cell membrane voltage changes, cell membrane conductivitychanges, a kinase activity triggered upon binding of a compound to thepolypeptide or complex, generation, breakdown or binding of a phorbolester by the polypeptide or complex, binding of diacylglycerol or otherlipids by the polypeptide or complex, cAMP activity, cGMP activity,GTPgammaS binding, phospholipase C activity, activity of an enzymeinvolved in cellular ionic balance, binding of polycystin-2L1orpolycystin-1L3 to each other or the relevant polypeptide or complex toanother polycystin-type protein, or a transcriptional reporter activityassay, e.g., using CRE, SRE, MRE, TRE, NFAT, and/or NFkB-responseelements coupled to appropriate reporters.

In one embodiment, cell-free assays for identifying such compoundscomprise a reaction mixture containing a polycystin polypeptide orcomplex encoded by PKD2-L1 and/or PKD1-L3 and a test compound or alibrary of test compounds. Accordingly, one example of a cell-freemethod for identifying test compounds that specifically bind to PC-1-L3,PC-2-L1, or PC-1-L3/PC-2-L1 complexes comprises contacting a PC-1-L3protein, PC-2-L1 protein, or PC-1-L3/PC-2-L1 protein complex, orfunctional fragment thereof, with a test compound or library of testcompounds and detecting the formation of test-compound/protein complexesby conventional methods. Similarly, the effect on PC-1-L3/PC-2-L1complex formation by the test compound can also be determined bymonitoring association of the polycystin proteins in the presence andabsence of the test compound.

In one class of useful embodiments, a library of the test compounds canbe synthesized on a solid substrate, e.g., a solid surface, plastic pinsor some other surface. The test compounds are reacted with thepolycystin polypeptide(s) and washed to elute unbound polypeptide(s).Bound polypeptide is then detected by methods well known in the art. Areciprocal assay can also be used, e.g., in which polypeptide (ormembrane-associated polypeptide) is applied directly onto plates andbinding of the test compound to the polypeptide is detected. Antibody orligand binding to the polypeptide or complexes can also be detected ineither format.

Interaction between molecules can also be assessed using real-time BIA(Biomolecular Interaction Analysis, e.g., using devices from PharmaciaBiosensor AB), which detect surface plasmon resonance (an opticalphenomenon). Detection depends on changes in the mass concentration ofmacromolecules at the biospecific interface and does not requirespecific labeling of the molecules. In one useful embodiment, a libraryof test compounds can be immobilized on a sensor surface, e.g., a wallof a micro-flow cell. A solution containing the PC-1-L3 and/or PC-2-L1polypeptide or complex is then continuously circulated over the sensorsurface. An alteration in the resonance angle, as indicated on a signalrecording, indicates the occurrence of an interaction. This generaltechnique is described in more detail in the BIAtechnology Handbook byPharmacia.

Optionally, the PC-1-L3 and/or PC-2-L1 polypeptide(s) is/are immobilizedto facilitate separation of complexes between the polypeptide(s) and atest compound from uncomplexed forms of the polypeptide(s). This alsofacilitates automation of the assay. Complexation of polypeptide(s) witheach other and/or the test compound can be achieved in any type ofvessel, e.g., microtitre plates, microfluidic chambers or channels,microcentrifuge tubes and test tubes. In one embodiment, the PC-1-L3and/or PC-2-L1 polypeptide is fused to another protein, e.g.,glutathione-5-transferase to form a fusion protein which can be adsorbedonto a matrix, e.g., glutathione Sepharose™ beads (Sigma Chemical. St.Louis, Mo.), which are then combined with the test compound andincubated under conditions sufficient to form complexes. Subsequently,the beads are washed to remove unbound label, and the matrix isimmobilized and the radiolabel is determined.

Similar methods for immobilizing proteins on matrices use biotin andstreptavidin. For example, the proteins or complexes can be biotinylatedusing biotin NHS (N-hydroxy-succinimide), using well known techniquesand immobilized in the well of streptavidin-coated plates.

Cell-free assays can also be used to identify tastants or othercompounds (e.g., potential pH response modulators) that bind and/ormodulate the activity of a PC-1-L3, PC-2-L1 or PC-1-L3/PC-2-L1polypeptide or complex. In one embodiment, the polypeptide or complex isincubated with a test compound and a transmembrane ion channel activityof the polypeptide or complex is determined. In another embodiment, thebinding affinity of the polypeptide or complex to a target molecule isdetermined by standard methods.

Further Details Regarding Cell Based Assays

In addition to cell-free assays such as those described above, thePC-1-L3 and/or PC-2-L1 polypeptide, and/or complex thereof can be usedin cell-based assay for identifying compounds that bind to, activateand/or modulate polypeptide or complex activity.

For example, one method for identifying compounds that bind to PC-1-L3and/or PC-2-L1 polypeptides or complexes comprises providing a cell thatexpresses one or both of these proteins, e.g., a human PC-1-L3 and/orPC-2-L1 polypeptide, combining a test compound with the cell andmeasuring the formation of a complex between the test compound and thepolypeptide or polypeptide complex. The cell can be a mammalian cell(e.g., a CHO cell), a yeast cell, a bacterial cell, an insect cell, aXenopus oocyte, a human or other mammalian taste cell, a kidney cell orany other cell expressing the PC-1-L3 and/or PC-2-L1 polypeptide,whether that expression is natural to the cell or the result ofrecombinant introduction of a PKD1L3 and/or PKD2L1 gene of interest.Further details regarding appropriate cells is found below.

In another embodiment, taste cells, kidney cells, neuronal cells, orcells expressing heterologous PC-1-L3 or PC-2-L1 polypeptides orcomplexes (e.g., recombinant CHO cells, recombinant insect cells,recombinant human cell line cells, etc.) or membrane preparations ofsuch cells, can be utilized to screen for bioactivity of test compounds.The PC-2-L1 polypeptides described herein are Ca²⁺ permeable cationselective channels (pore forming channels). G-proteins such as PC-1-L3interact with PC-2 proteins such as PC-2-L1. A variety of intracellulareffectors have been identified as being Ca²⁺/G-protein regulatedincluding, but not limited to, Ca²⁺-induced intraorganellar Ca²⁺ releaseby ryanodine and/or IP3 receptors, adenyl cyclase, cyclic GMP,phospholipase C, phospholipase A2 and phosphodiesterases, etc.Accordingly, the level of such second messengers produced by theaforementioned intracellular effectors, and thus activity of PC-1-L3and/or PC-2-L1 polypeptides and/or polypeptide complexes, can bemeasured by techniques that are well known.

For example, the level of cAMP produced by activation of adenyl cyclasecan be measured by assays which monitor cAMP, either in vivo by usingFRET or transcriptional reporters sensitive to cAMP, or in vitro bydirectly measuring cAMP production. The GTPase activity by G proteinssuch as PC-1-L3 can be measured, e.g., in plasma membrane preparationsby measuring the hydrolysis of gamma ³²P GTP, or in vivo by FRET or bymonitoring activity of downstream effectors such as PLC, adenylatecyclase, etc. Breakdown of phosphatidylinositol-4,5-bisphosphate to1,4,5-IP3 and diacylglycerol can be monitored by measuring the amount ofdiacylglycerol using thin-layer chromatography, or measuring the amountof IP3 using radiolabeling techniques or HPLC, or in vivo by activationof the IP3 receptor and release of calcium from internal stores. Thegeneration of arachidonic acid by the activation of phospholipase A2 canbe readily quantitated by well-known techniques.

Efflux of intracellular calcium or influx of calcium from outside thecell can be measured using conventional techniques, e.g., loading cellswith a Ca⁺ sensitive fluorescent dye such as fura-2 or indol-1, andmeasuring any change in Ca⁺⁺ using a fluorometer, such as FluoskanAscent Fluorescent Plate Reader or Fluorometric Imaging Plate Reader.The signal pathways initiated by PC-1-L3 and/or PC-2-L1 polypeptides orpolypeptide complexes in response to sour, acid, base or other compoundscan also be monitored by reporter gene assays.

Assays that monitor changes in membrane potential by (1) voltagemeasurements in Xenopus oocytes injected with mRNA encoding PC-1-L3 orPC-2-L1, (2) patch clamping in tissue culture cells expressing thereceptor, and (3) fluorometric assays using voltage-sensitive dyes orionic fluxes are preferred assays for monitoring membrane potential inthe context of the present invention.

In other aspects, interactions between PC-1-L3, PC-2-L1 and/or relatedproteins (e.g., other polycystins) are monitored to detect activity orbinding properties of the polypeptides or complexes thereof. Forexample, PC-2-like proteins (which are typically ion channels) ofteninteract with PC-1-like proteins (which are typically G-proteins) toprovide functional receptor complexes. Thus, in one aspect, interactionsbetween polycystin-2L1 and polycystin-1L3 can be monitored. In addition,homodimers and heterodimers between different PC-1 and PC-2 proteins canexist. Accordingly, binding between PC-1-L3, PC-2-L1 and/or otherpolycystins can be monitored, e.g., by FRET or other protein-proteininteraction technologies (cross-linking, etc.) to monitor homodimer andheterodimer formation, gating by PC-1 type or PC-2 type or relatedproteins, or the like.

As described, other assays such as melanophore assays, Phospholipase Cassays, Ca⁺⁺ mobilization assays, beta-arrestin FRET assays, andtranscriptional reporter assays, e.g., using CRE, SRE, MRE, TRE, NFAT,and/or NFkB-response elements coupled to appropriate reporters can beused. Detection using reporter genes coupled to appropriate responseelements are particularly convenient. For example, the coding sequenceto chloramphenicol acetyl transferase, beta galactosidase or otherconvenient markers are coupled to a response element that is activatedby a second messenger that is activated by a protein of the invention,e.g., through Ca⁺⁺ release. Cells expressing the marker in response toapplication of an appropriate test compound are detected by cellsurvival, or by expression of a calorimetric marker, or the like,according to well established methods.

Any of a variety of potential modulators of PC-1-L3, PC-2-L1,PC-1-L3/PC-2-L1 or PKD1L3/PKD2L1 activity or expression can be screenedfor. For example, potential modulators (acids, bases, ions, sour/acidsubstitutes, small organic molecules, peptides, peptide mimetics, weakacids, CO₂, acetic acid, blockers of carbonic anhydrase, MK-417, smallmolecules, organic molecules, inorganic molecules, proteins, hormones,transcription factors, or the like) can be contacted to a cell and aneffect on PC-1-L3 and/or PC-2-L1 polypeptide(s) or complexes and/orPKD1L3 or PKD2L1 activity and/or expression monitored by any of theassays described herein or known in the art.

Furthermore, expression of PKD1L3 and/or PKD2L1 can be detected, e.g.,via northern analysis or quantitative (e.g., real time) RT-PCR, beforeand/or after application of potential expression modulators. Similarly,promoter regions of PKD1L3 and/or PKD2L1 gene(s) of interest (e.g.,generally sequences in the region of the start site of transcription,e.g., within 5 KB of the start site, e.g., 1 KB, or less e.g., within500 BP or 250 BP or 100 BP of the start site) can be coupled to reporterconstructs (CAT, beta-galactosidase, luciferase or any other availablereporter) and can be similarly be tested for expression activitymodulation by the potential modulator. In either case, the assays can beperformed in a high-throughput fashion, e.g., using automated fluidhandling and/or detection systems, in a serial or parallel fashion.Similarly, activity modulators can be tested by contacting a potentialmodulator to an appropriate cell using any of the activity detectionmethods herein, regardless of whether the activity that is detected isthe result of activity modulation, expression modulation or both.

In any of the assays herein, control compounds can be administered andthe activity of the control compounds compared to those of the testcompounds to verify that changes in activity resulting from applicationof the test compound are not artifacts. For example, control compoundscan include the various dyes, buffers, adjuvants, carriers, or the likethat the test compounds are typically administered with, but lacking aputative test compound.

Details Regarding Transmembrane Potential Measurements and TransmembraneDyes

As noted above, the invention optionally includes monitoringtransmembrane potential (TM potential) to track ion channel activity ofPC-1-13, PC-2-L1 or complexes thereof. In general, the distribution of apermeable ion between the inside and outside of a cell or other membranedepends on the transmembrane potential of the cell membrane. Inparticular, for ions separated by a semi-permeable membrane, theelectrochemical potential difference (Δμ_(j)) which exists across themembrane, is given by Δμ_(j)=2.3 RT log [j_(I)]/[j_(o)]+zE_(R)F, where Ris the universal gas constant, T is an absolute temperature of thecomposition, F is Faraday's constant in coulombs, [j_(I)] is theconcentration of an ion (j) on an internal or intracellular side of theat least one membrane, [j_(o)] is the concentration of j on an externalor extracellular side of the at least one membrane, z is a valence of jand E_(R) is a measured transmembrane potential. Thus, the calculatedequilibrium potential difference (E_(j)) for ion j=−2.3RT(zF)⁻¹ log[j_(I)]/[j_(o)] (this is often referred to as the “Nernst equation”).See, Selkurt, ed. (1984) Physiology 5^(th) Edition, Chapters 1 and 2,Little, Brown, Boston, Mass. (ISBN 0-316-78038-3); Stryer (1995)Biochemistry 4^(th) edition Chapters 11 and 12, W.H. Freeman andCompany, NY (ISBN 0-7167-2009-4); Haugland (1996) Handbook ofFluorescent Probes and Research Chemicals Sixth Edition by MolecularProbes, Inc. (Eugene Oreg.) Chapter 25 (Molecular Probes, 1996) andhttp://www.probes.com/handbook/sections/2300.html (Chapter 23 of theon-line 1999 version of the Handbook of Fluorescent Probes and ResearchChemicals Sixth Edition by Molecular Probes, Inc.) (Molecular Probes,1999) and ille (1992) Ionic Channels of Excitable Membranes, secondedition, Sinauer Associates Inc. Sunderland, Mass. (ISBN 0-87893-323-9)(Hille), for an introduction to transmembrane potential and theapplication of the Nernst equation to transmembrane potential. Inaddition to the Nernst equation, various calculations which factor inthe membrane permeability of an ion, as well as Ohm's law, can be usedto further refine the model of transmembrane potential difference, suchas the “Goldman” or “constant field” equation and Gibbs-Donnanequilibrium. See Selkurt, ed. (1984) Physiology 5^(th) Edition, Chapter1, Little, Brown, Boston, Mass. (ISBN 0-3,6-78038-3) and Hille at e.g.,chapters 10-13.

Increases and decreases in resting transmembrane potential—referred toas membrane depolarization and hyperpolarization, respectively—play acentral role in many physiological processes, including ion-channelgating. Potentiometric optical probes (typically potentiometric dyes)provide a tool for measuring transmembrane potential and changes intransmembrane potential over time (e.g., transmembrane potentialresponses following the addition of a composition which affectstransmembrane potential) in membrane containing structures such asorganelles, cells and in vitro membrane preparations. In conjunctionwith probe imaging techniques (e.g., visualization of the relevantdyes), dye probes are used to map variations in transmembrane potentialacross cells membranes.

Potentiometric probes include cationic or zwitterionic styryl dyes,cationic rhodamines, anionic oxonols, hybrid oxonols and merocyanine540. The class of dye determines factors such as accumulation in cells,response mechanism and cell toxicity. See, Molecular Probes 1999 and thereference cited therein; Plasek et al. (1996) “Indicators ofTransmembrane potential: a Survey of Different Approaches to ProbeResponse Analysis.” J Photochem Photobiol; Loew (1994) “Characterizationof Potentiometric Membrane Dyes.” Adv Chem Ser 235, 151 (1994); Wu andCohen (1993) “Fast Multisite Optical Measurement of Transmembranepotential” Fluorescent and Luminescent Probes for Biological Activity,Mason, Ed., pp. 389-404; Loew (1993) “Potentiometric Membrane Dyes.”Fluorescent and Luminescent Probes for Biological Activity, Mason, Ed.,pp. 150-160; Smith (1990) “Potential-Sensitive Molecular Probes inMembranes of Bioenergetic Relevance.” Biochim Biophys Acta 1016, 1;Gross and Loew (1989) “Fluorescent Indicators of Transmembranepotential: Microspectrofluorometry and Imaging.” Meth Cell Biol 30, 193;Freedman and Novak (1989) “Optical Measurement of Transmembranepotential in Cells, Organelles, and Vesicles” Meth Enzymol 172, 102(1989); Wilson and Chused (1985) “Lymphocyte Transmembrane potential andCa⁺²-Sensitive Potassium Channels Described by Oxonol Dye FluorescenceMeasurements” Journal of Cellular Physiology 125:72-81; Epps et al.(1993) “Characterization of the Steady State and Dynamic FluorescenceProperties of the Potential Sensitive dye bis-(1,3-dibutylbarbituricacid) trimethine oxonol (DiBAC₄(3) in model systems and cells” Chemistryof Physics and Lipids 69:137-150, and Tanner et al. (1993) “FlowCytometric Analysis of Altered Mononuclear Cell Transmembrane potentialInduced by Cyclosporin” Cytometry 14:59-69.

Potentiometric dyes are typically divided into at least two categoriesbased on their response mechanism. The first class of dyes, referred toas fast-response dyes (e.g., styrylpyridinium dyes; see, e.g., MolecularProbes (1999) at Section 23.2), operate by a change in the electronicstructure of the dye, and consequently the fluorescence properties ofthe dye, i.e., in response to a change in an electric field whichsurrounds the dye. Optical response of these dyes is sufficiently fastto detect transient (millisecond) potential changes in excitable cells,e.g., isolated neurons, cardiac cells, and even intact brains. Themagnitude of the potential-dependent fluorescence change is often small;fast-response probes typically show a 2-10% fluorescence change per 100mV.

The second class of dyes, referred to as slow-response (or “Nernstian”)dyes (See, e.g., Molecular Probes, 1999 at Section 23.3), exhibitpotential-dependent changes in membrane distribution that areaccompanied by a fluorescence change. The magnitude of their opticalresponses is typically larger than that of fast-response probes.Slow-response probes, which include cationic carbocyanines, rhodaminesand anionic oxonols, are suitable for detecting changes in a variety oftransmembrane potentials of, e.g., nonexcitable cells caused by avariety of biological phenomena, including ion channel permeability. Thestructures of a variety of available slow response dyes are found e.g.,at table 25.3 of Molecular Probes (1996).

Many slow, Nemstian dyes such as carbocyanines, rhodamines and oxonolsare used to measure transmembrane potential by virtue ofvoltage-dependent dye redistribution and fluorescence changes resultingfrom the redistribution. Fluorescence changes which may be caused byredistribution include: a change of the concentration of the fluorophorewithin the cell or vesicle, a change in the dye fluorescence due toaggregation or a change in dye fluorescence due to binding tointracellular or intravesicular sites. Typically, 10-15 minutes ofequilibration time is used to allow the dyes to redistribute across thecell membrane after changing the transmembrane potential.

Examples of available anionic dyes that can be used for measurement oftransmembrane potential include the anionic bis-isoxazolone oxonolswhich accumulate in the cytoplasm of depolarized cells by a Nernstequilibrium-dependent uptake from the extracellular solution. Of theoxonols studied in one reference (“Kinetics of the Potential-SensitiveExtrinsic Probe Oxonol VI in Beef Heart Submitochondrial Particles.” J.C. Smith, B. Chance. J Membrane Biol 46, 255 (1979)), oxonol VI gave thelargest spectral shifts, with an isosbestic point at 603 nm. Oxonol VIresponds to changes in potential more rapidly than oxonol V.

The three common bis-barbituric acid oxonols, often referred to as DiBACdyes, form a family of spectrally distinct potentiometric probes withexcitation maxima at approximately 490 nm (DiBAC₄(3), 530 nm(DiSBAC₂(3)) and 590 nm (DiBAC₄(5)). DiBAC₄(3) has been used in manypublications that cite using a “bis-oxonol” (Molecular Probes, 1999,chapter 23). The dyes enter depolarized cells where they bind tointracellular proteins or membranes and exhibit enhanced fluorescenceand red spectral shifts. Increased depolarization results in more influxof the anionic dye and thus an increase in fluorescence. DiBAC₄(3) hasparticularly high voltage sensitivity. The long-wavelength DiSBAC₂(3)has frequently been used in combination with the UV light-excitable Ca²⁺indicators indo-1 or fura-2 for the simultaneous measurements oftransmembrane potential and Ca²⁺ concentrations (id. at Table 23.2).

Classes of cationic membrane permeable dyes that can be used as ionsensing compositions include, e.g., indo-carbocyanine dyes,thio-carbocyanine dyes, oxa-carbocyanine dyes (see Molecular Probeson-line catalogue, updated as of Aug. 10, 2000, at section 23.3,entitled “Slow-Response Dyes;”http://www.probes.com/handbook/sections/2303.html). See also, Sims, etal. (1974) “Studies on the Mechanism by Which Cyanine Dyes MeasureMembrane Potential in Red Blood Cells and Phosphatidylcholine Vesicles,”Biochemistry 13, 3315; Cabrini and Verkman (1986) “Potential-SensitiveResponse Mechanism of DiS-C3(5) in Biological Membranes,” Membrane Biol92, 171; Guillet and Kimmich (1981) “DiO-C3-(5) and DiS-C3-(5):Interactions with RBC, Ghosts and Phospholipid Vesicles,” J MembraneBiol 59, 1; Rottenberg and Wu (1998) “Quantitative Assay by FlowCytometry of the Mitochondrial Membrane Potential in Intact Cells,”Biochim Biophys Acta 1404, 393 (1998).

Other useful transmembrane dyes include amino napthylethylenylpyridinium dyes, and dialkyl amino phenyl polyphenyl pyridinium dyes.The amino napthylethylenyl pyridinium dyes include the ANEP type dyes,e.g., listed in the Molecular Probes catalog (Di-4-ANEPPS, Di-8-ANEPPS,Di-2-ANEPEQ, Di-8-ANEPEQ and Di-12-ANEPEQ). Dialkyl amino phenylpolyphenyl pyridinium dyes include the RH type dyes listed in theMolecular Probes catalog (RH160, RH237, RH 421, RH 704, RH 414, and RH461).

In general, changes in the level of fluorescence of the biologicalsample (e.g., containing PC-1-L3 and/or PC-2-L1 and/or coding nucleicacids)-test compound mixture are detected, where the change influorescence is indicative of a change in transmembrane potential.Typically, the assay methods described herein are used to detect theeffect of the test compound on the transmembrane potential of a cell orother membrane. Where one is seeking to determine the effect of a testcompound on a cell's transmembrane potential, e.g., through a change inion flux, transport, membrane permeability, or the like, one can exposethe cell, membrane, etc., to the test compound and the cell etc., isexamined for the presence of a previously absent fluorescent signal (orthe absence of a previously present fluorescent signal). Of particularinterest are the effects of tastant compounds and potential modulatorson cellular functioning, as determinable from TMP measurements.

For example, in one assay format, a dye is contacted to a biologicalsample. In accordance with these methods, the sample can be placed intoa reaction vessel, such as a microwell dish, and the level offluorescence from the composition is measured, optionally over a periodof time. This can be used to provide an initial or background level offluorescence indicative of an existing transmembrane potential for thebiological sample. A selected test compound is then added to thebiological sample (or these procedures are carried out in parallel,providing control and experimental samples). The test compound can betested alone, or is added before, together or after addition of tastantsto determine its effect on tastant responses (e.g. enhancement orinhibition). Following the stimulus, the fluorescence level of thebiological sample is again measured (typically over time) and comparedto the initial fluorescent level or the fluorescence level in a controlcell population (e.g., which is exposed to a control TMP modulator). Anychange in the level of fluorescence not attributable to dilution by thetest compound (as determined from an appropriate control) is thenattributable to the effect the test compound has on the cell'stransmembrane potential, or rate of TMP change in response todepolarization or hyperpolarization events.

A suitable negative control can be used in the assay, such as abiological sample that does not include the PC-1-L3 and/or PC-2-L1and/or coding nucleic acid(s), to ensure that the effect being observedis caused by the relevant protein or complex. Similarly, a suitablepositive control can be used in the assay, such as a test compound knownto effect the protein, gene or complex under study, to ensure that thebiological sample components are suitably active.

In any case, these types of assays are optionally carried out in anappropriate reaction receptacle that allows measurement of fluorescence,in situ. As such, the receptacle is typically a transparent reactionvessel, such as a test tube, cuvette, a reaction well in a multiwellplate, or a transparent conduit, e.g., a capillary, microchannel ortube.

The assay methods of the present invention are particularly useful inperforming high-throughput (greater than 1,000 compounds/day) and evenultra-high throughput (e.g., greater than 10,000 compounds/day)screening of chemical libraries, e.g., in searching fortastant/modulator leads. These experiments may be carried out inparallel by a providing a large number of reaction mixtures (e.g., cellsuspensions as described herein) in separate receptacles, typically in amultiwell format, e.g., 96 well, 324 well or 1536 well plates. Differenttest compounds (library members) are added to separate wells, and theeffect of the compound on the reaction mixture is ascertained, e.g., viathe fluorescent signal. These parallelized assays are generally carriedout using specialized equipment e.g., as described above to enablesimultaneous processing of large numbers of samples, i.e., fluidhandling by robotic pipettor systems and fluorescent detection bymultiplexed fluorescent multi-well plate readers.

Patch Clamping

As noted above, monitoring of transmembrane dye flow is a preferredmethod of monitoring test compound effects on ion channels. A secondpreferred method uses voltage clamping, such as patch clamping. This isa particularly useful method e.g., when using Xenopus oocytes.

A voltage clamp allows for the measurement of ion currents flowingacross a cell membrane. Originally, the voltage clamp used twoelectrodes and a feedback circuit for transmembrane measurements. In theoriginal Cole-Marmount voltage clamp, both electrodes are placed insidea cell and transmembrane voltage is recorded through one of theelectrodes (the “voltage electrode”) relative to an outside reference(e.g., ground). The second electrode passes current into the cell and istermed the “current electrode”.

Briefly, a “holding voltage” is maintained across the cell membrane.Anytime the cell makes a deviation from this holding voltage by passingan ion current across its membrane, an operational amplifier generatesan “error signal”. The error signal is the difference between theholding voltage specified by the experimenter and the actual voltage ofthe cell. The feedback circuit of the voltage clamp passes current intothe cell (via the current electrode) in the polarity needed to reducethe error signal to zero. Thus, the current is applied in a polarityopposite current that the cell is passing across its membrane, and theclamp circuit provides a current that is the mirror image of thecellular current. This mirror or “clamp current” can be easily measured,giving an accurate reproduction of the currents flowing across thecell's membrane (although in the opposite polarity).

A modem variant of this general method is the “patch clamp” which uses asingle electrode device. The patch clamp technique is in common use tomonitor the flow of ions across a membrane (Neher E (1992) “Nobellecture. Ion channels for communication between and within cells”Neuron. 8(4):605-12). The patch clamp technique involves applying a veryfinely drawn glass micropipette onto the surface of a cell to form anelectrode. This electrode is pressed against a cell membrane and suctionis applied to the inside of the electrode to pull the cell's membraneinside the tip of the electrode. This suction causes the cell to form atight seal with the electrode (a “giga-ohm seal,” as the electricalresistance of the seal is in excess of one giga-ohm). From this point,at least 4 different experimental approaches can be taken. First, theelectrode can be left sealed to a patch of membrane (a “cell-attachedpatch”). This allows for the recording of currents through single ionchannels in that patch of membrane. Second, the electrode can bewithdrawn from the cell, ripping a patch of membrane off of the cell.This forms an “inside-out” patch. This is useful when the environment onthe inside of an ion channel is to be studied. Third, the electrode canbe withdrawn from the cell, allowing a blob of membrane to bud from thecell. When the electrode is pulled away, this blob will part from thecell and reform as a ball of membrane on the end of the electrode, withthe outside of the membrane being the surface of the ball (thus the name“outside out patch”). Such “outside out” patching permits examination ofthe properties of an ion channel when it is protected from the outsideenvironment, but not in contact with it's usual environment. Fourth, theelectrode can be left in place, but harder suction is applied to rupturethe portion of the cell's membrane that is inside the electrode,providing access to the intracellular space of the cell. This is knownas “whole-cell recording”. This method is also sometimes misnamed a“whole cell patch.” The advantage of whole cell recording is that thesum total current that flows across the cell's membrane can be recorded.

Thus, the voltage clamping such as the patch clamp technique allows therecording of single ion-channel currents, or alternatively currents fromentire small cells. In the context of the present invention, thisprovides a platform for the analysis of changes in currents that resultfrom application of a test compound of modulator.

A modern variant of the classical patch clamp that can be adapted to thepresent invention is the planar patch clamp, which uses a planar arrayof PDMS electrodes that mimic a classical glass electrode (Klemic et al.(2002) “Micromolded PDMS Electrode Allows Patch Clamp ElectricalRecording From Cells” Biosensors and Bioelectronics 597-604). Thismodern patch clamp is suited to high throughput patch clamp analysis,allowing many different cells to be analyzed for ion channel activitysimultaneously. Patch clamp devices are also commercially available,e.g., from Axon Instruments.

Additional Screening System Details

Automated systems of the invention can facilitate the screening methodsnoted above (both in vitro and in vivo screening methods). That is,systems that facilitate cell or biochemical sample based screening forPC-1-L3, PC-2-L1, PKD1L3, or PKD2L1 expression and/or activity are afeature of the invention. Similarly, systems designed to monitorfeeding/drinking/licking etc. behavior of animals, or physiologicalresponses of animals (respiration rate, oxygen consumption, blood pH,etc.), including non-human transgenic laboratory animals, are also afeature of the invention. System features herein are generallyapplicable to the methods herein and vice-versa.

Biological/Biochemical Sources/Libraries

High-throughput automated systems that detect compounds that bind toand/or modulate an activity of PC-1-L3, or PC-2-L1 receptorpolypeptide(s), or complex(es) thereof, typically include abiological/biochemical sample (that includes the taste receptorpolypeptide or complex, e.g., any cell or other material describedherein) and a source of a plurality of test compounds. A detectordetects binding of one or more of the test compounds to the polypeptideor complex, or modulation of a level (e.g., complex formation) oractivity of the polypeptide or complex (or mRNA transcript(s)corresponding to the polypeptide(s)) by the test compounds, therebyidentifying a putative modulator, tastant compound, acid receptorbinding moiety, etc., that binds to or modulates the activity of thepolypeptide or complex.

The source of test compound for such systems and in the practice of themethods of the invention can be any commercially available orproprietary library of materials, including compound libraries fromSenomyx (La Jolla, Calif.), Sigma (St. Louis Mo.), Aldrich (St. LouisMo.), Agilent Technologies (Palo Alto, Calif.) or the like. The formatof the library will vary depending on the system to be used. In onetypical embodiment, libraries of sample materials are arrayed inmicrowell plates (e.g., 96, 384 or more well plates), which can beaccessed by standard fluid handling robotics, e.g., using a pipettor orother fluid handler with a standard ORCA robot (Optimized Robot forChemical Analysis) available from Beclaman Coulter (Fullerton, Calif.).Standard commercially available workstations such as the Caliper LifeSciences (Hopkinton, Mass.) Sciclone ALH 3000 workstation andRapidplatem™ 96/384 workstation provide precise 96 and 384-well fluidtransfers in a small, highly scalable format. Plate management systemssuch as the Caliper Life Sciences Twister® II Advanced CapabilityMicroplate Handler for End-Users, OEM's and Integrators provide platehandling, storage and management capabilities for fluid handling, whilethe Presto™ AutoStack provides fast reliable access to consumablespresenting trays of tips, reagents, microplates or deep wells to anautomated device (e.g., the ALH 3000) without robotic arm intervention.

Microfluidic systems for handling and analyzing microscale fluidsamples, including cell based and non-cell based approaches that can beused for analysis of test compounds on biological samples in the presentinvention are also available, e.g., the Caliper Life Sciences variousLabChip® technologies (e.g., LabChip® 90 and 3000) and related AgilentTechnologies (Palo Alto, Calif.) 2100 and 5100 devices. Similarly,interface devices between microfluidic and standard plate handlingtechnologies are also commercially available. For example, the CaliperTechnologies LabChip® 3000 uses “sipper chips” as a “chip-to-world”interface that allows automated sampling from microtiter plates. To meetthe needs of high-throughput environments, the LabChip® 3000 employsfour or even twelve sippers on a single chip so that samples can beprocessed, in parallel, up to twelve at a time. Solid phase libraries ofmaterials can also be conveniently accessed using sipper or pipettingtechnology, e.g., solid phase libraries can be gridded on a surface anddried for later rehydration with a sipper or pipette and accessedthrough the sipper or pipette.

As already noted, with regard to the systems and methods of theinvention, the particular libraries of compounds can be any of thosethat now exist, e.g., those that are commercially available, or that areproprietary. A number of libraries of test compounds exist, e.g., thosefrom Senomyx (La Jolla, Calif.) (which include libraries pre-screenedfor desirable tastant properties), Sigma (St. Louis Mo.), and Aldrich(St. Louis Mo.). Other current compound library providers includeActimol (Newark Del.), providing e.g., the Actiprobe 10 and Actiprobe 25libraries of 10,000 and 25,000 compounds, respectively; BioMol(Philedelphia, Pa.), providing a variety of libraries, including naturalcompound libraries and the Screen-Well™ Ion Channel ligand library whichare usefully screened against the receptors herein, as well as severalother application specific libraries; Enamine (Kiev, Ukranie) whichproduces custom libraries of billions of compounds from thousands ofdifferent building blocks, TimTec (Newark Del.), which produces generalscreening stock compound libraries containing >100,000 compounds, aswell as template-based libraries with common heterocyclic lattices,libraries for targeted mechanism based selections, including kinasemodulators, GPCR Ligands, channel modulators, etc., privileged structurelibraries that include compounds containing chemical motifs that aremore frequently associated with higher biological activity than otherstructures, diversity libraries that include compounds pre-selected fromavailable stocks of compounds with maximum chemical diversity, plantextract libraries, natural products and natural product-derivedlibraries, etc; AnalytiCon Discovery (Germany) including NatDiverse(natural product analogue screening compounds) and MEGAbolite (naturalproduct screening compounds); Chembridge (San Diego, Calif.) including awide array of targeted or general and custom or stock libraries; ChemDiv(San Diego, Calif.) providing a variety of compound diversity librariesincluding CombiLab and the International Diversity Collection; Comgenix(Hungary) including ActiVerse™ libraries; MicroSource (Gaylordsville,Conn.) including natural libraries, agro libraries, the NINDS customlibrary, the genesis plus library and others; Polyphor (Switzerland)including privileged core structures as well as novel scaffolds;Prestwick Chemical (Washington D.C.), including the Prestwick chemicalcollection and others that are pre-screened for biotolerance; Tripos(St. Louis, Mo.), including large lead screening libraries; and manyothers. Academic institutions such as the Zelinsky Institute of OrganicChemistry (Russian Federation) also provide libraries of considerablestructural diversity that can be screened in the methods of theinvention.

Detectors and Other System Components

Although the devices and systems specifically illustrated herein aregenerally described in terms of the performance of a few or oneparticular operation, it will be readily appreciated from thisdisclosure that these systems permit easy integration of additionaloperations. For example, the systems described will optionally includestructures, reagents and systems for performing virtually any number ofoperations both upstream and downstream from the operations specificallydescribed herein. Such upstream operations include sample handling andpreparation operations, e.g., cell separation, extraction, purification,culture, amplification, cellular activation, labeling reactions,dilution, aliquotting, and the like. Similarly, downstream operationsmay include similar operations, including, e.g., separation of samplecomponents, labeling of components, assays and detection operations,movement of components into contact with cells or other membranepreparations, or materials released from cells or membrane preparations,or the like.

Upstream and downstream assay and detection operations include, withoutlimitation, cell fluorescence assays, cell activity assays,receptor/ligand assays, immunoassays, and the like. Any of theseelements can be fixed incorporated into the systems herein.

Instrumentation for high throughput optical screening of cell assays isavailable. In addition to the systems noted herein, other automatedapproaches can also be practiced with the dyes and methods of theinvention. For example, the FLIPR (Fluorescence Imaging Plate Reader)was developed to perform quantitative optical screening for cell basedkinetic assays (Schroder and Neagle (1996) “FLIPR: A New Instrument forAccurate, High Throughput Optical Screening” Journal of BiomolecularScreening 1(2):75-80). This device can be adapted to the presentinvention, e.g., by using dyes to monitor TMP, as discussed above.

In general in the present invention, materials such as cells and dyesare optionally monitored and/or detected so that an activity such as TMPactivity can be determined. Depending on the label signal measurements,decisions can be made regarding subsequent operations, e.g., whether toassay a particular tastant/modulator in detail to determine detailedreceptor binding/activity kinetic information.

The systems described herein generally include fluid handling devices,as described above, in conjunction with additional instrumentation forcontrolling fluid transport, flow rate and direction within the devices,detection instrumentation for detecting or sensing results of theoperations performed by the system, processors, e.g., computers, forinstructing the controlling instrumentation in accordance withpreprogrammed instructions, receiving data from the detectioninstrumentation, and for analyzing, storing and interpreting the data,and providing the data and interpretations in a readily accessiblereporting format. Patch clamps, or other features described herein arealso optionally features of the invention.

Controllers

A variety of controlling instrumentation is optionally utilized inconjunction with the fluid handling elements described above, forcontrolling the transport and direction of fluids and/or materials(biological samples, test compounds, etc.) within the systems of thepresent invention. Controllers typically include appropriate softwaredirecting fluid and material transport in response to user instructions.

Typically, the controller systems are appropriately configured toreceive or interface with a fluid handling or other system element asdescribed herein. For example, the controller and/or detector,optionally includes a stage upon which a sample is mounted to facilitateappropriate interfacing between the controller and/or detector and therest of the system. Typically, the stage includes an appropriatemounting/alignment structural elements, such as a nesting well,alignment pins and/or holes, asymmetric edge structures (e.g., tofacilitate proper alignment of slides, microwell plates or microfluidic“chips”), and the like.

Detectors

Within the systems, detectors can take any of a variety of forms. Thevarious fluid handling stations noted above often come with integrateddetectors, e.g., optical or fluorescent detectors. However, otherdetectors such as patch clamp devices, fluorescence detectors thatdetects FRET, changes in membrane potential or flow of a dye into or outof the cell are also suitable, depending on the application.

Generally, devices herein optionally include signal detectors, e.g.,which detect fluorescence, phosphorescence, radioactivity, pH, charge,absorbance, luminescence, temperature, magnetism or the like. As noted,fluorescent and patch clamp detection is especially preferred andgenerally used for detection of voltage changes, or flow of voltagesensitive compounds (however, as noted, upstream and downstreamoperations can be performed on cells, dyes, modulators or the like,which can involve other detection methods).

System signal detectors are typically disposed adjacent to a site ofreaction or mixing between the biological/biochemical sample and thetest compound. This site can be a test tube, microwell plate,microfluidic device, or the like. The site is within sensorycommunication of the detector. The phrase “within sensory communication”generally refers to the relative location of the detector that ispositioned relative to the site so as to be able to receive a particularrelevant signal from that container. In the case of optical detectors,e.g., fluorescence FRET, or fluorescence polarization detectors, sensorycommunication typically means that the detector is disposed sufficientlyproximal to the container that optical, e.g., fluorescent signals, aretransmitted to the detector for adequate detection of those signals.Typically this employs a lens, optical train or other detection element,e.g., a CCD, that is focused upon a relevant portion of the container toefficiently gather and record these optical signals.

Example detectors include patch-clamp stations, photo multiplier tubes,spectrophotometers, a CCD array, a scanning detector, a microscope, agalvo-scann or the like. Cells, dyes or other components which emit adetectable signal can be flowed past or moved into contact with thedetector, or, alternatively, the detector can move relative to an arrayof samples (or, the detector can simultaneously monitor a number ofspatial positions corresponding to samples, e.g., as in a CCD array).

The system typically includes a signal detector located proximal to thesite of mixing/reaction. The signal detector detects the detectablesignal, e.g., for a selected length of time (t). For example, thedetector can include a spectrophotometer, or an optical detectionelement. Commonly, the signal detector is operably coupled to acomputer, which deconvolves the detectable signal to provide anindication of the transmembrane potential, e.g., an indication of achange in the potential over time.

The detector can detect transmembrane potential (the work needed to movea unit of charge across a membrane such as a cell membrane), e.g.,through a patch clamp, or by detecting flow of a cationic membranepermeable dye, an anionic Nernstian dye, an anionic membrane permeabledye, or other voltage sensing composition across the membrane over time,e.g., in response to application of a test compound. Changes in the rateof depolarization and hyperpolarization are monitored in response to atest compound, e.g., as compared to a control that does not include thetest compound. Permeable dyes are particularly useful for monitoring ionflow, e.g., dyes that can equilibrate across the membrane relativelyquickly, typically in about 1 hour, or less. Permeability can bedependent upon the relevant conditions, e.g., temperature, ionicconditions, voltage potentials, or the like.

Computer

Either or both of the controller system and/or the detection system areoptionally coupled to an appropriately programmed processor or computerwhich functions to instruct the operation of these instruments inaccordance with preprogrammed or user input instructions, receive dataand information from these instruments, and interpret, manipulate andreport this information to the user. As such, the computer is typicallyappropriately coupled to one or both of these instruments (e.g.,including an analog to digital or digital to analog converter asneeded).

The computer typically includes appropriate software for receiving userinstructions, either in the form of user input into a set parameterfields, e.g., in a GUI, or in the form of preprogrammed instructions,e.g., preprogrammed for a variety of different specific operations. Thesoftware then converts these instructions to appropriate language forinstructing the operation of the fluid direction and transportcontroller to carry out the desired operation. The computer thenreceives the data from the one or more sensors/detectors included withinthe system, and interprets the data, either provides it in a userunderstood format, or uses that data to initiate further controllerinstructions, in accordance with the programming, e.g., such as inmonitoring and control of flow rates, temperatures, applied voltages,and the like.

In the present invention, the computer typically includes software forthe monitoring of samples. Additionally, the software is optionally usedto control flow of materials.

Biosensors

Biosensors of the invention are devices or systems that comprisePC-1-L3, PC-2-L1 and/or complexes thereof, coupled to a readout thatmeasures or displays one or more activity of the polypeptide. Thus, anyof the above described assay components can be configured as a biosensorby operably coupling the appropriate assay components to a readout. Thereadout can be optical (e.g., to detect cell markers, ion-sensitivedyes, cell potential, or cell survival) electrical (e.g., coupled to aPET, a BIAcore, or any of a variety of others), spectrographic, or thelike, and can optionally include a user-viewable display (e.g., a CRT oroptical viewing station). The biosensor can be coupled to robotics orother automation, e.g., microfluidic systems, that direct contact of thetest compounds to the proteins of the invention, e.g., for automatedhigh-throughput analysis of test compound activity. A large variety ofautomated systems that can be adapted to use with the biosensors of theinvention are commercially available. For example, automated systemshave been made to assess a variety of biological phenomena, including,e.g., expression levels of genes in response to selected stimuli(Service (1998) “Microchips Arrays Put DNA on the Spot” Science282:396-399). Laboratory systems can also perform, e.g., repetitivefluid handling operations (e.g., pipetting) for transferring material toor from reagent storage systems that comprise arrays, such as microtitertrays or other chip trays, which are used as basic container elementsfor a variety of automated laboratory methods. Similarly, the systemsmanipulate, e.g., microtiter trays and control a variety ofenvironmental conditions such as temperature, exposure to light or air,and the like. Many such automated systems are commercially available.Examples of automated systems are available from Caliper Technologies(including the former Zymark Corporation, Hopkinton, Mass.), whichutilize various Zymate systems which typically include, e.g., roboticsand fluid handling modules. Similarly, the common ORCA® robot, which isused in a variety of laboratory systems, e.g., for microtiter traymanipulation, is also commercially available, e.g., from BeckmanCoulter, Inc. (Fullerton, Calif.). A number of automated approaches tohigh-throughput activity screening are provided by the GenomicsInstitute of the Novartis Foundation (La Jolla, Calif.); See GNF.org onthe world-wide web. Microfluidic screening applications are alsocommercially available from Caliper Technologies Corp. For example,(e.g., LabMicrofluidic Device® high throughput screening system (HTS) byCaliper Technologies, Mountain View, Calif. or the HP/Agilenttechnologies Bioanalyzer using LabChip™ technology by CaliperTechnologies Corp. can be adapted for use in the present invention.

In an alternate embodiment, conformational changes are detected bycoupling the relevant polypeptide(s) or complex(es) to an electricalreadout, e.g., to a chemically coupled field effect transistor (aCHEM-FET) or other appropriate system for detecting changes inconductance or other electrical properties brought about by aconformational shift by the protein of the invention.

Further Details Regarding Methods of Monitoring PC-1-L3 and/or PC-2-L1Induced Behavior in Animal Models

In addition to the various biological and biochemical sample-basedscreening methods noted herein, the invention also encompasses testingfor PC-1-L3/PKDL3 and/or PC-2-L1/PKD2L1 activity in response to testcompounds, in vivo. In one embodiment, this is accomplished byintroducing a heterologous PKD1-L3 and/or PKD2L1 taste receptor geneinto an animal and expressing an encoded heterologous PC-1-L3 and/orPC-2-L1 taste receptor polypeptide in a taste bud of the animal or otherrelevant target for cellular expression (e.g., optionally including anycell or tissue type that naturally expresses PKD2L1 and/or PKD1L3). Aputative PC-1-L3 and/or PC-2-L1 taste receptor tastant or modulator isprovided to the animal, and one or more feeding behavior orphysiological response of the animal is monitored in response to thepresence of the putative PC-1-L3 and/or PC-2-L1 taste receptor tastant.

Optionally, the animal is a knock-out animal that has a reduced oreliminated function of an endogenous PKD1L3 and/or PKD2L1 gene and/orencoded polypeptide(s), e.g., in taste bud cells. Knock out animals areuseful both for studies of PC-1-L3/PKDL3 and/or PC-2-L1/PKD2L1 function(for example, conformation that an animal lacking PC-1-L3/PKDL3 and/orPC-2-L1/PKD2L1 is deficient with respect to one or more tasteperceptions or physiological, e.g., pH monitoring, responses) and as atarget for delivery of a heterologous PKDL3 and/or PKD2L1 gene. That is,in one aspect, the animal is made transgenic with a PKD1L3 and/or PKD2L1gene of interest. For example, a PKD1L3 and/or PKD2L1 knock-out mousethat comprises a human transgene for human PKD1L3 and/or PKD2L1 willdisplay a response to tastants and modulators similar to a human,providing a good model system for studying response to tastants andmodulators. Double knock outs of PKD2L1 and PKD1L3 are useful forproviding both genes from a heterologous (e.g., human) source. Theheterologous gene can be placed under the control of a heterologouspromoter that is active in taste bud cells, e.g., a PKD1L3 and/or PKD2L1promoter, a T1R-gene promoter, T2R-gene promoter, TRPM5-gene promoter, aPLCB2 gene promoter, a repeater gene promoter, a gustducin genepromoter, a Gi2 gene promoter, a cytokeratin-19 gene promoter, oranother promoter for a gene that is naturally selectively expressed in ataste receptor cell of the tongue or palate epithelium.

Feeding behavior or physiological response(s) of the animal in responseto putative tastants and/or modulators can be monitored by availablemethods. For example, animals (such as a transgenic PKD1L3 and/or PKD2L1knock out mouse or other configuration as noted above that comprises ahuman PKD1L3 and/or PKD2L1 gene) will lick a device (stick, tube, plate,etc.) coated with a tastant, if the tastant is perceived as pleasurableto the animal. By monitoring increased or decreased licking behavior onsuch devices, the effect of a putative tastant or modulator on feedingbehavior can be determined. Similarly, a putative tastant or modulatorcan be dissolved in a taste neutral fluid such as water and supplied tothe animal (e.g., using a water bottle) to determine if drinkingbehavior increases, or if the fluid with the putative tastant is drunkpreferentially to the neutral fluid (or avoided). For example, aneutrally flavored “control” can be a water bottle, while a testcompound flavored “experimental” bottle can be placed in a controlbottle. If the animal (mouse, rabbit, rat, etc.) feeds preferentially onthe experimental bottle, then the animal can taste the test compound andperceives the flavor as pleasurable. If the experimental and controlbottle are drunk equally, then the animal likely cannot taste the testcompound. If the experimental bottle is drunk less than the control,then the animal can likely detect the test compound, and may detect itas being unpleasant. Similar experiments can be performed with a foodsource flavored with the test compound. Physiological responses that canbe monitored in such animals also can include respiration rate, oxygenconsumption, blood or urine pH, or the like. Measuring these responsesutilizes standard techniques.

Modulatory activity can be similarly determined. That is, a potentialmodulator can be administered to the animal (e.g., applied to the tastebud, injected, or supplied in food or drink) and the increase ordecrease in feeding/drinking/licking behavior towards a known tastant(e.g., a sour tastant) can be detected, and/or a physiological responsecan be detected, essentially as above. If administration of themodulator results in an increase in feeding/drinling/licling behaviortowards the known tastant, then the modulator potentiates the responseof that taste quality. If feeding/drinking/licking is decreased, then itlikely inhibits activity of an attractive taste modality, or enhancesactivity of an aversive taste modality. Either activity can be useful,depending on whether an increase in feeding/drinking is desirable, e.g.,to reduce adverse flavor effects of acid preservatives, or a decrease infeeding/drinking is desirable (e.g., to treat obesity, metabolicsyndrome, high blood pressure, or the like by reducing calorieconsumption). Examples of modulators include taste or pH receptoragonists, enhancers, antagonists, inverse agonists, etc.

Behavioral Systems

As noted, a further aspect of the invention monitors animal behaviorupon application of potential tastants or taste cell modulators. Thesesystems include a non-human animal comprising a heterologous PKD1L3and/or PKD2L1 taste receptor gene that is expressed in a taste bud ofthe animal and a source of a putative tastant or modulator that isaccessible (or administratable) to the animal. The system furtherincludes a detector that detects a feeding behavior of the animal inresponse to the presence of the putative tastant or modulator.

Here again, the animal is typically a knock-out animal (e.g., a mouse)deficient in endogenous PC-1-L3 and/or PC-2-L1 taste receptorpolypeptide expression, that expresses a heterologous human PC-1-L3and/or PC-2-L1 taste receptor polypeptide. The source can include any ofthe configurations noted above with respect to the related methods,e.g., a lickable device, a fluid source comprising the tastant, or afood source comprising the tastant.

The detector will typically include a camera or motion sensor thatmonitors movement of the animal. Alternately, lickable devices candetect pressure against the device through conventional strainmeasurement devices, or electronically by detecting the completion of acircuit upon licking, or optically by detecting tongue movement. It isalso possible to inset electrodes in muscles controlling oromotoractivity and monitor their contraction/relaxation as a surrogate forfeeding and gagging behavior.

An analysis module, e.g., a computer analyzes information from thedetector and can statistically compile information regardingfeeding/licking/drinking behavior. The analysis module can include auser viewable display that displays the results of the analysis to auser, e.g., a GUI.

Making Knock-Out Animals and Transgenics

Transgenic animals are a useful tool for studying gene function andtesting tastants and modulators. Human (or other selected) PKD1L3 and/orPKD2L1 genes can be introduced in place of endogenous PKD1L3 and/orPKD2L1 genes of a laboratory animal, making it possible to studyfunction of the human (or other) taste receptor in the easilymanipulated and studied laboratory animal. It will be appreciated thatthere is not precise correspondence between receptor function ofdifferent animals (humans and mice perceive aspartame differently, forexample), making the ability to study the human or other receptor ofinterest particularly useful. Although similar genetic manipulations canbe performed in tissue culture, the interaction of PKD1L3 and/or PKD2L1and/or PC-1-L3 and/or PC-2-L1 and/or complexes thereof, in the contextof an intact organism, provides a more complete and physiologicallyrelevant picture of PKD1L3/PKD2L1/PC-1-L3/PC-2-L1 function than can beachieved in simple cell-based screening assays. Accordingly, one featureof the invention is the creation of transgenic animals comprisingheterologous PKD1L3 and/or PKD2L1 genes, e.g., which express these genesin the taste buds of the transgenic animal.

In general, such a transgenic animal is typically an animal that has hadappropriate PKD1L3 and/or PKD9L2 genes (or partial genes, e.g.,comprising coding sequences coupled to a promoter) introduced into oneor more of its cells artificially. This is most commonly done in one oftwo ways. First, a DNA encoding PC-1-L3 and/or PC-2-L1 (or fragmentsthereof) can be integrated randomly by injecting it into the pronucleusof a fertilized ovum. In this case, the DNA can integrate anywhere inthe genome. In this approach, there is no need for homology between theinjected DNA and the host genome. Second, targeted insertion can beaccomplished by introducing the (heterologous) DNA into embryonic stem(ES) cells and selecting for cells in which the heterologous DNA hasundergone homologous recombination with homologous sequences of thecellular genome. Typically, there are several kilobases of homologybetween the heterologous and genomic DNA, and positive selectablemarkers (e.g., antibiotic resistance genes) are included in theheterologous DNA to provide for selection of transformants. In addition,negative selectable markers (e.g., “toxic” genes such as barnase) can beused to select against cells that have incorporated DNA bynon-homologous recombination (i.e., random insertion).

One common use of targeted insertion of DNA is to make knock-out mice.Typically, homologous recombination is used to insert a selectable genedriven by a constitutive promoter into an essential exon of the genethat one wishes to disrupt (e.g., the first coding exon). To accomplishthis, the selectable marker is flanked by large stretches of DNA thatmatch the genomic sequences surrounding the desired insertion point.Once this construct is electroporated into ES cells, the cells' ownmachinery performs the homologous recombination. To make it possible toselect against ES cells that incorporate DNA by non-homologousrecombination, it is common for targeting constructs to include anegatively selectable gene outside the region intended to undergorecombination (typically the gene is cloned adjacent to the shorter ofthe two regions of genomic homology). Because DNA lying outside theregions of genomic homology is lost during homologous recombination,cells undergoing homologous recombination cannot be selected against,whereas cells undergoing random integration of DNA often can. A commonlyused gene for negative selection is the herpes virus thymidine kinasegene, which confers sensitivity to the drug gancyclovir.

Following positive selection and negative selection if desired, ES cellclones are screened for incorporation of the construct into the correctgenomic locus. Typically, one designs a targeting construct so that aband normally seen on a Southern blot or following PCR amplificationbecomes replaced by a band of a predicted size when homologousrecombination occurs. Since ES cells are diploid, only one allele isusually altered by the recombination event so, when appropriatetargeting has occurred, one usually sees bands representing both wildtype and targeted alleles.

The embryonic stem (ES) cells that are used for targeted insertion arederived from the inner cell masses of blastocysts (early mouse embryos).These cells are pluripotent, meaning they can develop into any type oftissue.

Once positive ES clones have been grown up and frozen, the production oftransgenic animals can begin. Donor females are mated, blastocysts areharvested, and several ES cells are injected into each blastocyst.Blastocysts are then implanted into a uterine horn of each recipient. Bychoosing an appropriate donor strain, the detection of chimericoffspring (i.e., those in which some fraction of tissue is derived fromthe transgenic ES cells) can be as simple as observing hair and/or eyecolor. If the transgenic ES cells do not contribute to the germline(sperm or eggs), the transgene cannot be passed on to offspring.

Further Details Regarding Cells Comprising PKD1L3/PKD2L1/PC-1-L3/PC-2-L1

As already noted, for several embodiments, biological samples to betested for PKD1L3/PKD2L1 expression or PC-1-L3/PC-2-L1 expression orconcentration are cells or are derived from cell preparations. The cellscan be those associated with PKD1L3 and/or PKD2L1 and/or PC-1-L3 and/orPC-2-L1 expression in vivo, such as taste bud, nerve or kidney cells.Alternately, the cells can be derived from a taste bud, nerve or kidneycell, e.g., through culture.

However, one feature of the invention is the production of recombinantcells, e.g., expressing a heterologous PKD1L3 and/or PKD2L1 gene. It isworth noting that recombinant cells expressing both recombinant PKD1L3and PKD2L1 are a feature of the invention that arises out of thedetermination that both genes are expressed in taste bud cells, whichwas not previously known.

In these recombinant cell embodiments, the biological sample to betested is derived from the recombinant cell, which is selected largelyfor ease of culture and manipulation. The cells can be, e.g., human,rodent, insect, Xenopus, etc. and will typically be a cell in culture(or an oocyte in the case of Xenopus).

PKD1L3 and/or PKD2L1 nucleic acids are typically introduced into cellsin cloning and/or expression vectors to facilitate introduction of thenucleic acid and expression of PKD1L3 and/or PKD2L1 to produce PC-1-L3and/or PC-2-L1. Vectors include, e.g., plasmids, cosmids, viruses, YACs,bacteria, poly-lysine, etc. A “vector nucleic acid” is a nucleic acidmolecule into which a heterologous nucleic acid is optionally insertedthat can then be introduced into an appropriate host cell. Vectorspreferably have one or more origins of replication, and one or moresites into which the recombinant DNA can be inserted. Vectors often haveconvenient means by which cells with vectors can be selected from thosewithout, e.g., they encode drug resistance genes. Common vectors includeplasmids, viral genomes, and (primarily in yeast and bacteria)artificial chromosomes. “Expression vectors” are vectors that compriseelements that provide for or facilitate transcription of nucleic acidswhich are cloned into the vectors. Such elements can include, e.g.,promoters and/or enhancers operably coupled to a nucleic acid ofinterest.

In general, appropriate expression vectors are known in the art. Forexample, pET-14b, pcDNA1Amp, and pVL1392 are available from Novagen andInvitrogen and are suitable vectors for expression in E. coli, COS cellsand baculovirus infected insect cells, respectively. pcDNA-3, pEAK, andvectors that permit the generation of PKD2L1 RNA for in vitro and invivo expression experiments (e.g., in vitro translations and Xenopusoocyte injections) are also useful. These vectors are simplyillustrative of those that are known in the art, with thousands ofsuitable vectors being available. Suitable host cells can be any cellcapable of growth in a suitable media and allowing purification of anexpressed protein. Examples of suitable host cells include bacterialcells, such as E. coli, Streptococci, Staphylococci, Streptomyces andBacillus subtilis cells; fungal cells such as yeast cells, Pichia, andAspergillus cells; insect cells such as Drosophila S2 and Spodoptera Sf9cells, mammalian cells such as CHO, COS, and HeLa; and even plant cells.

Cells are transformed with PKD1L3 and/or PKD2L1 genes according tostandard cloning and transformation methods. PC-1-L3 and PC-2-L1 canalso be isolated from resulting recombinant cells using standardmethods. General texts which describe molecular biological techniquesfor making nucleic acids, including the use of vectors, promoters andmany other relevant topics, include Berger and Kimmel, Guide toMolecular Cloning Techniques, Methods in Enzymology volume 152 AcademicPress, Inc., San Diego, Calif. (Berger); Sambrook et al., MolecularCloning—A Laboratory Manual (3nd Ed.), Vol. 1-3, Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y., 2000 (“Sambrook”) and CurrentProtocols in Molecular Biology, F. M. Ausubel et al., eds., CurrentProtocols, a joint venture between Greene Publishing Associates, Inc.and John Wiley & Sons, Inc., (supplemented through 2002) (“Ausubel”)).

In addition, a plethora of kits are commercially available for thepreparation, purification and cloning of plasmids or other relevantnucleic acids from cells, (see, e.g., EasyPrep™, FlexiPrep™, both fromPharmacia Biotech; StrataClean™, from Stratagene; and, QIAprep™ fromQiagen). Any isolated and/or purified nucleic acid can be furthermanipulated to produce other nucleic acids, used to transfect cells,incorporated into related vectors to infect organisms, or the like.

As noted, typical vectors contain transcription and translationterminators, transcription and translation initiation sequences, andpromoters useful for regulation of the expression of the particulartarget nucleic acid. The vectors optionally comprise generic expressioncassettes containing at least one independent terminator sequence,sequences permitting replication of the cassette in eukaryotes, orprokaryotes, or both, (e.g., shuttle vectors) and selection markers forboth prokaryotic and eukaryotic systems. Vectors are suitable forreplication and integration in prokaryotes, eukaryotes, or both. See,Giliman & Smith, Gene 8:81 (1979); Roberts, et al., Nature, 328:731(1987); Schneider, B., et al., Protein Expr. Purif. 6435:10 (1995);Ausubel, Sambrook, Berger (above). A catalogue of Bacteria andBacteriophages useful for cloning is provided, e.g., by the ATCC, e.g.,The ATCC Catalogue of Bacteria and Bacteriophage published yearly by theATCC. Additional basic procedures for sequencing, cloning and otheraspects of molecular biology and underlying theoretical considerationsare also found in Watson et al. (1992) Recombinant DNA Second Edition,Scientific American Books, NY.

In addition, essentially any nucleic acid (and virtually any labelednucleic acid, whether standard or non-standard) can be custom orstandard ordered from any of a variety of commercial sources, such asThe Midland Certified Reagent Company (mcrc@oligos.com), The GreatAmerican Gene Company (www.genco.com), ExpressGen Inc.(www.expressgen.com), Operon Technologies Inc. (Alameda, Calif.) andmany others.

Other useful references, e.g. for cell isolation and culture (e.g., forsubsequent nucleic acid isolation) include Freshney (1994) Culture ofAnimal Cells, a Manual of Basic Technique, third edition, Wiley-Liss,New York and the references cited therein; Payne et al. (1992) PlantCell and Tissue Culture in Liquid Systems John Wiley & Sons, Inc. NewYork, N.Y.; Gamborg and Phillips (eds) (1995) Plant Cell, Tissue andOrgan Culture; Fundamental Methods Springer Lab Manual, Springer-Verlag(Berlin Heidelberg New York); and Atlas and Parks (eds) The Handbook ofMicrobiological Media (1993) CRC Press, Boca Raton, Fla.

Additional Details Regarding Protein Purification and Handling

Purification of PC-1-L3 and/or PC-2-L1, and/or complexes thereof can beaccomplished using known techniques. In one embodiment, transformedcells expressing PC-1-L3 and/or PC-2-L1 are lysed, crude purificationoccurs to remove debris and some contaminating proteins, followed bychromatography to further purify the protein to the desired level ofpurity. Because the proteins are membrane proteins, membrane fractionscomprising the proteins can similarly be purified, if desired. Cells canbe lysed by known techniques such as homogenization, sonication,detergent lysis and freeze-thaw techniques. Crude purification can occurusing ammonium sulfate precipitation, centrifugation or other knowntechniques. Suitable chromatography includes anion exchange, cationexchange, high performance liquid chromatography HPLC), gel filtration,affinity chromatography, hydrophobic interaction chromatography, etc.Well known techniques for refolding proteins can be used to obtain theactive conformation of the protein when the protein is denatured duringintracellular synthesis, isolation or purification.

In general, polycystin 2L1 polypeptides, can be purified, eitherpartially (e.g., achieving a 5×, 10×, 100×, 500×, or 1000× or greaterpurification), or even substantially to homogeneity (e.g., where theprotein is the main component of a solution, typically excluding thesolvent (e.g., water or DMSO) and buffer components (e.g., salts andstabilizers) that the polypeptide is suspended in, e.g., if thepolypeptide is in a liquid phase), according to standard proceduresknown to and used by those of skill in the art. Accordingly,polypeptides of the invention can be recovered and purified by any of anumber of methods well known in the art, including, e.g., ammoniumsulfate or ethanol precipitation, acid or base extraction, columnchromatography, affinity column chromatography, anion or cation exchangechromatography, phosphocellulose chromatography, hydrophobic interactionchromatography, hydroxylapatite chromatography, lectin chromatography,gel electrophoresis and the like. Protein refolding steps can be used,as desired, in making correctly folded mature proteins. High performanceliquid chromatography (HPLC), affinity chromatography or other suitablemethods can be employed in final purification steps where high purity isdesired. In one embodiment, antibodies made against polycyctin 2L1 areused as purification reagents, e.g., for affinity-based purification.Once purified, partially or to homogeneity, as desired, the polypeptidesare optionally used e.g., as assay components, therapeutic reagents oras immunogens for antibody production.

In addition to other references noted herein, a variety ofpurification/protein purification methods are well known in the art,including, e.g., those set forth in R. Scopes, Protein Purification,Springer-Verlag, N.Y. (1982); Deutscher, Methods in Enzmmology Vol. 182:Guide to Protein Purification, Academic Press, Inc. N.Y. (1990); Sandana(1997) Bioseparation of Proteins, Academic Press, Inc.; Bollag et al.(1996) Protein Methods, 2nd Edition Wiley-Liss, NY; Walker (1996) TheProtein Protocols Handbook Humana Press, NJ; Harris and Angal (1990)Protein Purification Applications: A Practical Approach IRL Press atOxford, Oxford, England; Harris and Angal Protein Purification Methods:A Practical Approach IRL Press at Oxford, Oxford, England; Scopes (1993)Protein Purification: Principles and Practice 3rd Edition SpringerVerlag, NY; Janson and Ryden (1998) Protein Purification: Principles,High Resolution Methods and Applications, Second Edition Wiley-VCH, NY;and Walker (1998) Protein Protocols on CD-ROM Humana Press, NJ; and thereferences cited therein.

Those of skill in the art will recognize that, after synthesis,expression and/or purification, proteins can possess a conformationdifferent from the desired conformations of the relevant polypeptides.For example, polypeptides produced by prokaryotic systems often areoptimized by exposure to chaotropic agents to achieve proper folding.During purification from, e.g., lysates derived from E. coli, theexpressed protein is optionally denatured and then renatured. This isaccomplished, e.g., by solubilizing the proteins in a chaotropic agentsuch as guanidine HCl. In general, it is occasionally desirable todenature and reduce expressed polypeptides and then to cause thepolypeptides to re-fold into the preferred conformation. For example,guanidine, urea, DTT, DTE, and/or a chaperonin can be added to atranslation product of interest. Methods of reducing, denaturing andrenaturing proteins are well known to those of skill in the art (see,the references above, and Debinski, et al. (1993) J. Biol. Chem., 268:14065-14070; Kreitman and Pastan (1993) Bioconjug. Chem., 4: 581-585;and Buchner, et al., (1992) Anal. Biochem., 205: 263-270). Debinski, etal., for example, describe the denaturation and reduction of inclusionbody proteins in guanidine-DTE. The proteins can be refolded in a redoxbuffer containing, e.g., oxidized glutathione and L-arginine. Refoldingreagents can be flowed or otherwise moved into contact with the one ormore polypeptide or other expression product, or vice-versa.

PKD1L3 and/or PKD2L1 nucleic acids optionally comprise a coding sequencefused in-frame to a marker sequence which, e.g., facilitatespurification of the encoded polypeptide. Such purification facilitatingdomains include, but are not limited to, metal chelating peptides suchas histidine-tryptophan modules that allow purification on immobilizedmetals, a sequence which binds glutathione (e.g., GST), a hemagglutinin(HA) tag (corresponding to an epitope derived from the influenzahemagglutinin protein; Wilson, I., et al. (1984) Cell 37:767), maltosebinding protein sequences, the FLAG epitope utilized in the FLAGSextension/affinity purification system (Immunex Corp, Seattle, Wash.),and the like. The inclusion of a protease-cleavable polypeptide linkersequence between the purification domain and the sequence of theinvention is useful to facilitate purification.

Cell Rescue—Treatement

In one aspect, the invention includes rescue of a cell that is defectivein function of one or more endogenous polycystin genes (e.g., PKD1L3and/or PKD2L1) or polypeptides (e.g., PC-1-L3 and/or PC-2-L1) orcomplexes thereof. This can be accomplished simply by introducing a newcopy of the gene(s) (or a heterologous nucleic acid(s) that expressesthe relevant protein(s)) into a cell. Other approaches, such ashomologous recombination to repair the defective gene (e.g., viachimeraplasty) can also be performed. In any event, rescue of functioncan be measured, e.g., in any of the assays noted herein. Indeed, thiscan be used as a general method of screening cells in vitro foractivity. Accordingly, in vitro rescue of function is useful in thiscontext for the myriad in vitro screening methods noted above, e.g., forthe identification of tastants or modulators in cells. The cells thatare rescued can include cells in culture, (including primary orsecondary cell culture from patients, as well as cultures ofwell-established cells). Where the cells are isolated from a patient,this has additional diagnostic utility in establishing which sequence isdefective in a patient that presents with a tasting defect.

In another aspect, cell rescue occurs in a patient, e.g., a human orveterinary patient, e.g., to remedy a tastant or pH sensor defect. Thus,one aspect of the invention is gene therapy to remedy tasting or pHsensing defects (or even simply to enhance tastant discrimination), inhuman or veterinary applications. In these applications, the nucleicacids of the invention are optionally cloned into appropriate genetherapy vectors (and/or are simply delivered as naked orliposome-conjugated nucleic acids), which are then delivered (generallytopically to the taste buds, but optionally systemically), optionally incombination with appropriate carriers or delivery agents. Proteins canalso be delivered directly, but delivery of the nucleic acid istypically preferred in applications where stable expression is desired.

Vectors for administration typically comprise PKD1L3 and/or PKD2L1 genesunder the control of a promoter that is expressed in taste bud cells.These can include native PKD1L3 or PKD2L1 promoters, or other taste budspecific promoters such as a T1R-gene promoter, a T2R— gene promoter, aTRPM5-gene promoter, a PLCB2 gene promoter, a repeater gene promoter, agustducin gene promoter, a Gi2 gene promoter, a cytokeratin-19 genepromoter, or a promoters for another gene that is naturally selectivelyexpressed in a taste receptor cell of the tongue or palate epithelium.In the case of expression in neuronal cells (e.g., in contact with theCSF), a variety of genes are known to be promiscuously expressed incentral or peripheral neurons. For example, Gray P A Fu H et al (2004)“Mouse Brain Organization Revealed through Direct Genome ScaleTranscription Factor Expression Analysis.” Science 306:2255-57 describegenes that can be used as sources of promoters. Similarly, Ruan et al(2005) “Nuclear receptors and their coregulators in kidney” Kidney Int.68(6):2444-61 describe appropriate sources of promoters for expressionin kidney.

Compositions for administration, e.g., comprise a therapeuticallyeffective amount of the gene therapy vector or other relevant nucleicacid, and a pharmaceutically acceptable carrier or excipient. Such acarrier or excipient includes, but is not limited to, saline, bufferedsaline, dextrose, water, glycerol, ethanol, and/or combinations thereof.The formulation is made to suit the mode of administration. In general,methods of administering gene therapy vectors for topical use are wellknown in the art and can be applied to administration of the nucleicacids of the invention.

Therapeutic compositions comprising one or more nucleic acid of theinvention are optionally tested in one or more appropriate in vitroand/or in vivo animal model of disease, to confirm efficacy, tissuemetabolism, and to estimate dosages, according to methods well known inthe art. In particular, dosages can initially be determined by activity,stability or other suitable measures of the formulation.

Administration is by any of the routes normally used for introducing amolecule into ultimate contact with cells of interest (taste bud,tongue, palate epithelium, neuronal cells, kidney cells, etc.).Practitioners can select an administration route of interest based onthe cell target. For example, topical administration or direct injectioninto the taste buds or other tissues of the tongue or palette epitheliumis simplest and therefore preferred for these targets. Similarly,injection into the CSF can be used where the target is neuronal cells incontact with the CSF. The nucleic acids of the invention areadministered in any suitable manner, optionally with one or morepharmaceutically acceptable carriers. Suitable methods of administeringsuch nucleic acids in the context of the present invention to a patientare available, and, although more than one route can be used toadminister a particular composition, a particular route can oftenprovide a more immediate and more effective action or reaction thananother route.

Pharmaceutically acceptable carriers are determined in part by theparticular composition being administered, as well as by the particularmethod used to administer the composition. Accordingly, there is a widevariety of suitable formulations of pharmaceutical compositions of thepresent invention. Compositions can be administered by a number ofroutes including, but not limited to: oral (in this case, topical andoral can be the same or different, e.g., topical delivery to the tastebuds can be oral, as can systemic administration by the GI tract),intravenous, intraperitoneal, intramuscular, transdermal, subcutaneous,topical, sublingual, spinal or rectal administration. Compositions canbe administered via liposomes (e.g., topically), or via topical deliveryof naked DNA or viral vectors. Such administration routes andappropriate formulations are generally known to those of skill in theart.

The compositions, alone or in combination with other suitablecomponents, can also be made into aerosol formulations (i.e., they canbe “nebulized”) to be administered via inhalation. Aerosol formulationscan be placed into pressurized acceptable propellants, such asdichlorodifluoromethane, propane, nitrogen, and the like. Formulationssuitable for parenteral administration, such as, for example, byintraarticular (in the joints), intravenous, intramuscular, intradermal,intraperitoneal, and subcutaneous routes, include aqueous andnon-aqueous, isotonic sterile injection solutions, which can containantioxidants, buffers, bacteriostats, and solutes that render theformulation isotonic with the blood of the intended recipient, andaqueous and non-aqueous sterile suspensions that can include suspendingagents, solubilizers, thickening agents, stabilizers, and preservatives.The formulations of packaged nucleic acid can be presented in unit-doseor multi-dose sealed containers, such as ampules and vials.

The dose administered to a patient, in the context of the presentinvention, is sufficient to effect a beneficial therapeutic response inthe patient over time, or, e.g., to provide sweet or glutamate tastantdiscrimination as perceived by the patient in an objective sweet orglutamate tastant test. The dose is determined by the efficacy of theparticular vector, or other formulation, and the activity, stability orserum half-life of the polypeptide which is expressed, and the conditionof the patient, as well as the body weight or surface area of thepatient to be treated. The size of the dose is also determined by theexistence, nature, and extent of any adverse side-effects that accompanythe administration of a particular vector, formulation, or the like in aparticular patient. In determining the effective amount of the vector orformulation to be administered in the treatment of disease, thephysician evaluates local expression in the taste buds, or circulatingplasma levels, formulation toxicities, progression of the relevantdisease, and/or where relevant, the production of antibodies to proteinsencoded by the polynucleotides. The dose administered, e.g., to a 70kilogram patient are typically in the range equivalent to dosages ofcurrently-used therapeutic proteins, adjusted for the altered activityor serum half-life of the relevant composition. The vectors of thisinvention can supplement treatment conditions by any known conventionaltherapy (e.g., diet restriction, etc.).

For administration, formulations of the present invention areadministered at a rate determined by the ILD-50 of the relevantformulation, and/or observation of any side-effects of the vectors ofthe invention at various concentrations, e.g., as applied to the mass ortopical delivery area and overall health of the patient. Administrationcan be accomplished via single or divided doses.

If a patient undergoing treatment develops fevers, chills, or muscleaches, he/she receives the appropriate dose of aspirin, ibuprofen,acetaminophen or other pain/fever controlling drug. Patients whoexperience reactions to the compositions, such as fever, muscle aches,and chills are premedicated 30 minutes prior to the future infusionswith either aspirin, acetaminophen, or, e.g., diphenhydramine.Meperidine is used for more severe chills and muscle aches that do notquickly respond to antipyretics and antihistainines. Treatment is slowedor discontinued depending upon the severity of the reaction.

Detecting Polymorphisms

In one aspect, the invention includes detecting a polymorphism in aPKD1L3 and/or PKD2L1 gene (or a nucleic acid in linkage disequilibriumwith such a polymorphism) to detect a taste receptor abnormality. A“polymorphism” is a locus that is variable; that is, within apopulation, the nucleotide sequence at a polymorphism has more than oneversion or allele. The term “allele” refers to one of two or moredifferent nucleotide sequences that occur or are encoded at a specificlocus, or two or more different polypeptide sequences encoded by such alocus. For example, a first allele can occur on one chromosome, while asecond allele occurs on a second homologous chromosome, e.g., as occursfor different chromosomes of a heterozygous individual, or betweendifferent homozygous or heterozygous individuals in a population. Oneexample of a polymorphism is a “single nucleotide polymorphism” (SNP),which is a polymorphism at a single nucleotide position in a genome (thenucleotide at the specified position varies between individuals orpopulations). An allele “positively” correlates with a trait when it islinked to it and when presence of the allele is an indictor that thetrait or trait form will occur in an individual comprising the allele.An allele negatively correlates with a trait when it is linked to it andwhen presence of the allele is an indicator that a trait or trait formwill not occur in an individual comprising the allele.

In the present case, the gene for tastant defects is identified (PKD1L3and/or PKD2L1). Polymorphisms within or linked to (in linkagedisequilibrium with) the gene likely correlate to altered tasteperception. Thus, tastant defects or abnormalities can be detected bydetecting polymorphisms in the gene.

In general, markers corresponding to polymorphisms between members of apopulation can be detected by numerous methods well-established in theart (e.g., PCR-based sequence specific amplification, restrictionfragment length polymorphisms (RFLPs), isozyme markers, northernanalysis, allele specific hybridization (ASH), array basedhybridization, amplified variable sequences of the genome,self-sustained sequence replication, simple sequence repeat (SSR),single nucleotide polymorphism (SNP), random amplified polymorphic DNA(“RAPD”) or amplified fragment length polymorphisms (AFLP). In oneadditional embodiment, the presence or absence of a molecular marker isdetermined simply through nucleotide sequencing of the polymorphicmarker region. Any of these methods are readily adapted to highthroughput analysis.

Additional Details Regarding Sequence Variations

A number of particular PC-1-L3 and PC-2-L1 polypeptides and codingnucleic acids are described herein by sequence (See, e.g., the Examplessection below; see also, co-pending application U.S. Ser. No.11/176,958). These polypeptides and coding nucleic acids can bemodified, e.g., by mutation as described herein, or simply by artificialsynthesis of a desired variant. Several types of example variants aredescribed below.

Splice Variants

Given the significant number of exons found in PKD1L3 and PKD2L1, thepresence of splice variants in taste receptor cells is likely. These canbe expressed alone or in combination and can be detected or monitored byanalysis of taste cell mRNA using PKD1L3 and/or PKD2L1 exon-specificprimers and the polymerase chain reaction.

Silent Variations

Due to the degeneracy of the genetic code, any of a variety of nucleicacids sequences encoding polypeptides of the invention are optionallyproduced, some which can bear lower levels of sequence identity to thePKD2L1 nucleic acids in the Examples below.

The following provides a typical codon table specifying the geneticcode, found in many biology and biochemistry texts.

TABLE 1 Codon Table Amino acids Codon Alanine Ala A GCA GCC GCG GCUCysteine Cys C UGC UGU Aspartic acid Asp D GAC GAU Glutamic acid Glu EGAA GAG Phenylalanine Phe F UUC UUU Glycine Gly G GGA GGC GGG GGUHistidine His H CAC CAU Isoleucine Ile I AUA AUC AUU Lysine Lys K AAAAAG Leucine Leu L UUA UUG CUA CUC CUG CUU Methionine Met M AUGAsparagine Asn N AAC AAU Proline Pro P CCA CCC CCG CCU Glutamine Gln QCAA CAG Arginine Arg R AGA AGG CGA CGC CGG CGU Serine Ser S AGC AGU UCAUCC UCG UCU Threonine Thr T ACA ACC ACG ACU Valine Val V GUA GUC GUG GUUTryptophan Trp W UGG Tyrosine Tyr Y UAC UAU

The codon table shows that many amino acids are encoded by more than onecodon. For example, the codons AGA, AGG, CGA, CGC, CGG, and CGU allencode the amino acid arginine. Thus, at every position in the nucleicacids of the invention where an arginine is specified by a codon, thecodon can be altered to any of the corresponding codons described abovewithout altering the encoded polypeptide. It is understood that U in anRNA sequence corresponds to T in a DNA sequence.

Such “silent variations” are one species of “conservatively modifiedvariations”, discussed below. One of skill will recognize that eachcodon in a nucleic acid (except ATG, which is ordinarily the only codonfor methionine) can be modified by standard techniques to encode afunctionally identical polypeptide. Accordingly, each silent variationof a nucleic acid which encodes a polypeptide is implicit in anydescribed sequence. The invention, therefore, explicitly provides eachand every possible variation of a nucleic acid sequence encoding apolypeptide of the invention that could be made by selectingcombinations based on possible codon choices. These combinations aremade in accordance with the standard triplet genetic code (e.g., as setforth in Table 1, or as is commonly available in the art) as applied tothe nucleic acid sequence encoding a polycystin polypeptide of theinvention. All such variations of every nucleic acid herein arespecifically provided and described by consideration of the sequence incombination with the genetic code. One of skill is fully able to makethese silent substitutions using the methods herein.

Conservative Variations

“Conservatively modified variations” or, simply, “conservativevariations” of a particular nucleic acid sequence or polypeptide arethose which encode identical or essentially identical amino acidsequences. One of skill will recognize that individual substitutions,deletions or additions which alter, add or delete a single amino acid ora small percentage of amino acids (typically less than 5%, moretypically less than 4%, 2% or 1%) in an encoded sequence are“conservatively modified variations” where the alterations result in thedeletion of an amino acid, addition of an amino acid, or substitution ofan amino acid with a chemically similar amino acid.

Conservative substitution tables providing functionally similar aminoacids are well known in the art. Table 2 sets forth six groups whichcontain amino acids that are “conservative substitutions” for oneanother.

TABLE 2 Conservative Substitution Groups 1 Alanine (A) Serine (S)Threonine (T) 2 Aspartic acid (D) Glutamic acid (E) 3 Asparagine (N)Glutamine (Q) 4 Arginine (R) Lysine (K) 5 Isoleucine (I) Leucine (L)Methionine (M) Valine (V) 6 Phenylalanine (F) Tyrosine (Y) Tryptophan(W)

Thus, “conservatively substituted variations” of a listed polypeptidesequence of the present invention include substitutions of a smallpercentage, typically less than 5%, more typically less than 2% or 1%,of the amino acids of the polypeptide sequence, with a conservativelyselected amino acid of the same conservative substitution group.

Finally, the addition or deletion of sequences which do not alter theencoded activity of a nucleic acid molecule, such as the addition ordeletion of a non-functional sequence (or, e.g., a tagging sequenceadded to facilitate purification), is a conservative variation of thebasic nucleic acid or polypeptide.

One of skill will appreciate that many conservative variations of thenucleic acid constructs which are disclosed yield a functionallyidentical construct. For example, as discussed above, owing to thedegeneracy of the genetic code, “silent substitutions” (i.e.,substitutions in a nucleic acid sequence which do not result in analteration in an encoded polypeptide) are an implied feature of everynucleic acid sequence which encodes an amino acid. Similarly,“conservative amino acid substitutions,” in one or a few amino acids inan amino acid sequence are substituted with different amino acids withhighly similar properties, are also readily identified as being highlysimilar to a disclosed construct. Such conservative variations of eachdisclosed sequence are a feature of the present invention.

Antibodies

In another aspect, antibodies to PC-1-L3 and/or PC-2-L1 and/or complexesthereof, can be generated using methods that are well known. Theantibodies can be utilized for detecting and/or purifying PC-1-L3 and/orPC-2-L1 and/or complexes thereof e.g., in situ to monitor localizationof receptor, or simply in a biological sample of interest. Antibodiescan optionally discriminate the PC-1-L3 and/or PC-2-L1 and/or complexesthereof from various other polycystin homologues, and/or can be used inbiosensor applications. Antibodies can also be used to block function ofpolycystin-2L1 and/or polycystin-1L3, and/or complexes thereof, in vivo,in situ or in vitro. As used herein, the term “antibody” includes, butis not limited to, polyclonal antibodies, monoclonal antibodies,humanized or chimeric antibodies and biologically functional antibodyfragments, which are those fragments sufficient for binding of theantibody fragment to the protein.

For the production of antibodies to a relevant polypeptide or complex,e.g., encoded by one of any disclosed or available sequences orconservative variant or fragment thereof for PKD1L3, PKD2L1, PC-1-L3 orPC-2-L1, various host animals maybe immunized by injection with thepolypeptide, or a portion thereof. Such host animals may include, butare not limited to, rabbits, mice and rats, to name but a few. Variousadjuvants may be used to enhance the immunological response, dependingon the host species, including, but not limited to, Freund's (completeand incomplete), mineral gels such as aluminum hydroxide, surface activesubstances such as lysolecithin, pluronic polyols, polyanions, peptides,oil emulsions, keyhole limpet hemocyanin, dinitrophenol, and potentiallyuseful human adjuvants such as BCG (bacille Calmette-Guerin) andCorynebacterium parvum.

Polyclonal antibodies are heterogeneous populations of antibodymolecules derived from the sera of animals immunized with an antigen,such as target gene product, or an antigenic functional derivativethereof. For the production of polyclonal antibodies, host animals, suchas those described above, may be immunized by injection with the encodedprotein, or a portion thereof, supplemented with adjuvants as alsodescribed above.

Monoclonal antibodies (mAbs), which are homogeneous populations ofantibodies to a particular antigen, may be obtained by any techniquewhich provides for the production of antibody molecules by continuouscell lines in culture. These include, but are not limited to, thehybridoma technique of Kohler and Milstein (Nature 256:495-497, 1975;and U.S. Pat. No. 4,376,110), the human B-cell hybridoma technique(Kosbor et al., Immunology Today 4:72, 1983; Cole et al., Proc. Nat'l.Acad. Sci. USA 80:2026-2030, 1983), and the EBV-hybridoma technique(Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss,Inc., pp. 77-96, 1985). Such antibodies may be of any immunoglobulinclass, including IgG, IgM, IgE, IgA, IgD, and any subclass thereof. Thehybridoma producing the mAb of this invention may be cultivated in vitroor in vivo. Production of high titers of mAbs in vivo makes this thepresently preferred method of production.

In addition, techniques developed for the production of “chimericantibodies” (Morrison et al., Proc. Nat'l. Acad. Sci. USA 81:6851-6855,1984; Neuberger et al., Nature 312:604-608, 1984; Takeda et al., Nature314:452-454, 1985) by splicing the genes from a mouse antibody moleculeof appropriate antigen specificity, together with genes from a humanantibody molecule of appropriate biological activity, can be used. Achimeric antibody is a molecule in which different portions are derivedfrom different animal species, such as those having a variable orhypervariable region derived from a murine mAb and a humanimmunoglobulin constant region.

Alternatively, techniques described for the production of single-chainantibodies (U.S. Pat. No. 4,946,778; Bird, Science 242:423-426, 1988;Huston et al., Proc. Nat'l. Acad. Sci. USA 85:5879-5883, 1988; and Wardet al., Nature 334:544-546, 1989) can be adapted to producedifferentially expressed gene-single chain antibodies. Single chainantibodies are formed by linking the heavy and light chain fragments ofthe Fv region via an amino acid bridge, resulting in a single-chainpolypeptide.

In one aspect, techniques useful for the production of “humanizedantibodies” can be adapted to produce antibodies to the proteins,fragments or derivatives thereof. Such techniques are disclosed in U.S.Pat. Nos. 5,932,448; 5,693,762; 5,693,761; 5,585,089; 5,530,101;5,569,825; 5,625,126; 5,633,425; 5,789,650; 5,661,016; and 5,770,429.

Antibody fragments which recognize specific epitopes may be generated byknown techniques. For example, such fragments include, but are notlimited to, the F(ab′)₂ fragments, which can be produced by pepsindigestion of the antibody molecule, and the Fab fragments, which can begenerated by reducing the disulfide bridges of the F(ab′)₂ fragments.Alternatively, Fab expression libraries may be constructed (Huse et al.,Science 246:1275-1281, 1989) to allow rapid and easy identification ofmonoclonal Fab fragments with the desired specificity.

The protocols for detecting and measuring the expression of thedescribed PC-1-L3 and/or PC-2-L1 and/or complexes herein, using theabove mentioned antibodies, are well known in the art. Such methodsinclude, but are not limited to, dot blotting, western blotting,competitive and noncompetitive protein binding assays, enzyme-linkedimmunosorbant assays (ELISA), immunohistochemistry,fluorescence-activated cell sorting (FACS), and others commonly used andwidely described in scientific and patent literature, and many employedcommercially.

One method, for ease of detection, is the sandwich ELISA, of which anumber of variations exist, all of which are intended to be encompassedby the present invention. For example, in a typical forward assay,unlabeled antibody is immobilized on a solid substrate and the sample tobe tested is brought into contact with the bound molecule and incubatedfor a period of time sufficient to allow formation of anantibody-antigen binary complex. At this point, a second antibody,labeled with a reporter molecule capable of inducing a detectablesignal, is then added and incubated, allowing time sufficient for theformation of a ternary complex of antibody-antigen-labeled antibody. Anyunreacted material is washed away, and the presence of the antigen isdetermined by observation of a signal, or may be quantitated bycomparing with a control sample containing known amounts of antigen.Variations on the forward assay include the simultaneous assay, in whichboth sample and antibody are added simultaneously to the bound antibody,or a reverse assay, in which the labeled antibody and sample to betested are first combined, incubated and added to the unlabeled surfacebound antibody. These techniques are well known to those skilled in theart, and the possibility of minor variations will be readily apparent.As used herein, “sandwich assay” is intended to encompass all variationson the basic two-site technique. For the immunoassays of the presentinvention, the only limiting factor is that the labeled antibody be anantibody which is specific for the protein expressed by the gene ofinterest.

The most commonly used reporter molecules in this type of assay areeither enzymes, fluorophore- or radionuclide-containing molecules. Inthe case of an enzyme immunoassay, an enzyme is conjugated to the secondantibody, usually by means of glutaraldehyde or periodate. As will bereadily recognized, however, a wide variety of different ligationtechniques exist which are well-known to the skilled artisan. Commonlyused enzymes include horseradish peroxidase, glucose oxidase,beta-galactosidase and alkaline phosphatase, among others. Thesubstrates to be used with the specific enzymes are generally chosen forthe production, upon hydrolysis by the corresponding enzyme, of adetectable color change. For example, p-nitrophenyl phosphate issuitable for use with alkaline phosphatase conjugates; for peroxidaseconjugates, 1,2-phenylenediamine or toluidine are commonly used. It isalso possible to employ fluorogenic substrates, which yield afluorescent product, rather than the chromogenic substrates noted above.A solution containing the appropriate substrate is then added to thetertiary complex. The substrate reacts with the enzyme linked to thesecond antibody, giving a qualitative visual signal, which may befurther quantitated, usually spectrophotometrically, to give anevaluation of the amount of PLAB which is present in the serum sample.

Alternately, fluorescent compounds, such as fluorescein and rhodamine,can be chemically coupled to antibodies without altering their bindingcapacity. When activated by illumination with light of a particularwavelength, the fluorochrome-labeled antibody absorbs the light energy,inducing a state of excitability in the molecule, followed by emissionof the light at a characteristic longer wavelength. The emission appearsas a characteristic color visually detectable with a light microscope.Immunofluorescence and EIA techniques are both very well established inthe art and are particularly preferred for the present method. However,other reporter molecules, such as radioisotopes, chemiluminescent orbioluminescent molecules may also be employed. It will be readilyapparent to the skilled artisan how to vary the procedure to suit therequired use.

In one example, peptides for PKD2L1 and PKD1L3 were generated, andconjugated to carriers. Rabbits were immunized to make polyclonalantibodies. These antibodies were shown to bind PC-2-L1 and PC-1-L3 insitu (see also, Example 3, herein).

Regulating Gene Expression of PKD2L1/PKD1L3

Gene expression (e.g., transcription and/or translation) of PKD2L1 orPKD1L3 can be regulated using any of a variety of techniques known inthe art. For example, gene expression can be inhibited using anantisense nucleic acid or an interfering RNA. Inhibition of expressionin particular cell-types can be used for further studying the in vitroor in vivo role of these genes, and/or as a mechanism for treating acondition caused by overexpression of a PKD1L3 or PKD2L1 gene, and/orfor treating a dominant effect caused by a particular allele of such agene (polycystic kidney disease is caused by such dominant alleles inrelated PKD genes).

For example, use of antisense nucleic acids is well known in the art. Anantisense nucleic acid has a region of complementarity to a targetnucleic acid, e.g., a target gene, mRNA, or cDNA. Typically, a nucleicacid comprising a nucleotide sequence in a complementary, antisenseorientation with respect to a coding (sense) sequence of an endogenousgene is introduced into a cell. The antisense nucleic acid can be RNA,DNA, a PNA or any other appropriate molecule. A duplex can form betweenthe antisense sequence and its complementary sense sequence, resultingin inactivation of the gene. The antisense nucleic acid can inhibit geneexpression by forming a duplex with an RNA transcribed from the gene, byforming a triplex with duplex DNA, etc. An antisense nucleic acid can beproduced, e.g., for any gene whose coding sequence is known or can bedetermined by a number of well-established techniques (e.g., chemicalsynthesis of an antisense RNA or oligonucleotide (optionally includingmodified nucleotides and/or linkages that increase resistance todegradation or improve cellular uptake) or in vitro transcription).Antisense nucleic acids and their use are described, e.g., in U.S. Pat.No. 6,242,258 to Haselton and Alexander (Jun. 5, 2001) entitled “Methodsfor the selective regulation of DNA and RNA transcription andtranslation by photoactivation”; U.S. Pat. No. 6,500,615; U.S. Pat. No.6,498,035; U.S. Pat. No. 6,395,544; U.S. Pat. No. 5,563,050; E. Schuchet al (1991) Symp Soc. Exp Biol 45:117-127; de Lange et al., (1995) CurrTop Microbiol Immunol 197:57-75; Hamilton et al., (1995) Curr TopMicrobiol Immunol 197:77-89; Finnegan et al., (1996) Proc Natl Acad SciUSA 93:8449-8454; Uhlmann and A. Pepan (1990), Chem. Rev. 90:543; P. D.Cook (1991), Anti-Cancer Drug Design 6:585; J. Goodchild, BioconjugateChem. 1 (1990) 165; and, S. L. Beaucage and R. P. Iyer (1993),Tetrahedron 49:6123; and F. Eckstein, Ed. (1991), Oligonucleotides andAnalogues—A Practical Approach, IRL Press.

Gene expression can also be inhibited by RNA silencing or interference.“RNA silencing” refers to any mechanism through which the presence of asingle-stranded or, typically, a double-stranded RNA in a cell resultsin inhibition of expression of a target gene comprising a sequenceidentical or nearly identical to that of the RNA, including, but notlimited to, RNA interference, repression of translation of a target mRNAtranscribed from the target gene without alteration of the mRNA'sstability, and transcriptional silencing (e.g., histone acetylation andheterochromatin formation leading to inhibition of transcription of thetarget mRNA).

The term “RNA interference” (“RNAi,” sometimes called RNA-mediatedinterference, post-transcriptional gene silencing, or quelling) refersto a phenomenon in which the presence of RNA, typically double-strandedRNA, in a cell results in inhibition of expression of a gene comprisinga sequence identical, or nearly identical, to that of thedouble-stranded RNA. The double-stranded RNA responsible for inducingRNAi is called an “interfering RNA.” Expression of the gene is inhibitedby the mechanism of RNAi as described below, in which the presence ofthe interfering RNA results in degradation of mRNA transcribed from thegene and thus in decreased levels of the mRNA and any encoded protein.

The mechanism of RNAi has been and is being extensively investigated ina number of eukaryotic organisms and cell types. See, for example, thefollowing reviews: McManus and Sharp (2002) “Gene silencing in mammalsby small interfering RNAs” Nature Reviews Genetics 3:737-747; Hutvagnerand Zamore (2002) “RNAi: Nature abhors a double strand” Curr Opin Genet& Dev 200:225-232; Hannon (2002) “RNA interference” Nature 418:244-251;Agami (2002) “RNAi and related mechanisms and their potential use fortherapy” Curr Opin Chem Biol 6:829-834; Tuschl and Borkhardt (2002)“Small interfering RNAs: A revolutionary tool for the analysis of genefunction and gene therapy” Molecular Interventions 2:158-167; Nishikura(2001) “A short primer on RNAi: RNA-directed RNA polymerase acts as akey catalyst” Cell 107:415-418; and Zamore (2001) “RNA interference:Listening to the sound of silence” Nature Structural Biology 8:746-750.RNAi is also described in the patent literature; see, e.g., CA 2359180by Kreutzer and Limmer entitled “Method and medicament for inhibitingthe expression of a given gene”; WO 01/68836 by Beach et al. entitled“Methods and compositions for RNA interference”; WO 01/70949 by Grahamet al. entitled “Genetic silencing”; and WO 01/75164 by Tuschl et al.entitled “RNA sequence-specific mediators of RNA interference.”

In brief, double-stranded RNA introduced into a cell (e.g., into thecytoplasm) is processed, for example by an RNAse III-like enzyme calledDicer, into shorter double-stranded fragments called small interferingRNAs (siRNAs, also called short interfering RNAs). The length and natureof the siRNAs produced is dependent on the species of the cell, althoughtypically siRNAs are 21-25 nucleotides long (e.g., an siRNA may have a19 base pair duplex portion with two nucleotide 3′ overhangs at eachend). Similar siRNAs can be produced in vitro (e.g., by chemicalsynthesis or in vitro transcription) and introduced into the cell toinduce RNAi. The siRNA becomes associated with an RNA-induced silencingcomplex (RISC). Separation of the sense and antisense strands of thesiRNA, and interaction of the siRNA antisense strand with its targetmRNA through complementary base-pairing interactions, optionally occurs.Finally, the mRNA is cleaved and degraded.

Expression of a target gene in a cell can thus be specifically inhibitedby introducing an appropriately chosen double-stranded RNA into thecell. Guidelines for design of suitable interfering RNAs are known tothose of skill in the art. For example, interfering RNAs are typicallydesigned against exon sequences, rather than introns or untranslatedregions. Characteristics of high efficiency interfering RNAs may vary bycell type. For example, although siRNAs may require 3′ overhangs and 5′phosphates for most efficient induction of RNAi in Drosophila cells, inmammalian cells blunt ended siRNAs and/or RNAs lacking 5′ phosphates caninduce RNAi as effectively as siRNAs with 3′ overhangs and/or 5′phosphates (see, e.g., Czaudema et al. (2003) “Structural variations andstabilizing modifications of synthetic siRNAs in mammalian cells” NuclAcids Res 31:2705-2716). As another example, since double-stranded RNAsgreater than 30-80 base pairs long activate the antiviral interferonresponse in mammalian cells and result in non-specific silencing,interfering RNAs for use in mammalian cells are typically less than 30base pairs (for example, Caplen et al. (2001) “Specific inhibition ofgene expression by small double-stranded RNAs in invertebrate andvertebrate systems” Proc. Natl. Acad. Sci. USA 98:9742-9747, Elbashir etal. (2001) “Duplexes of 21-nucleotide RNAs mediate RNA interference incultured mammalian cells” Nature 411:494-498 and Elbashir et al. (2002)“Analysis of gene function in somatic mammalian cells using smallinterfering RNAs” Methods 26:199-213 describe the use of 21 nucleotidesiRNAs to specifically inhibit gene expression in mammalian cell lines,and Kim et al. (2005) “Synthetic dsRNA Dicer substrates enhance RNAipotency and efficacy” Nature Biotechnology 23:222-226 describes use of25-30 nucleotide duplexes). The sense and antisense strands of a siRNAare typically, but not necessarily, completely complementary to eachother over the double-stranded region of the siRNA (excluding anyoverhangs). The antisense strand is typically completely complementaryto the target mRNA over the same region, although some nucleotidesubstitutions can be tolerated (e.g., a one or two nucleotide mismatchbetween the antisense strand and the mRNA can still result in RNAi,although at reduced efficiency). The ends of the double-stranded regionare typically more tolerant to substitution than the middle; forexample, as little as 15 bp (base pairs) of complementarity between theantisense strand and the target mRNA in the context of a 21 mer with a19 bp double-stranded region has been shown to result in a functionalsiRNA (see, e.g., Czauderna et al. (2003) “Structural variations andstabilizing modifications of synthetic siRNAs in mammalian cells” NuclAcids Res 31:2705-2716). Any overhangs can but need not be complementaryto the target mRNA; for example, TT (two 2′-deoxythymidines) overhangsare frequently used to reduce synthesis costs.

Although double-stranded RNAs (e.g., double-stranded siRNAs) wereinitially thought to be required to initiate RNAi, several recentreports indicate that the antisense strand of such siRNAs is sufficientto initiate RNAi. Single-stranded antisense siRNAs can initiate RNAithrough the same pathway as double-stranded siRNAs (as evidenced, forexample, by the appearance of specific mRNA endonucleolytic cleavagefragments). As for double-stranded interfering RNAs, characteristics ofhigh-efficiency single-stranded siRNAs may vary by cell type (e.g., a 5′phosphate may be required on the antisense strand for efficientinduction of RNAi in some cell types, while a free 5′ hydroxyl issufficient in other cell types capable of phosphorylating the hydroxyl).See, e.g., Martinez et al. (2002) “Single-stranded antisense siRNAsguide target RNA cleavage in RNAi” Cell 110:563-574; Amarzguioui et al.(2003) “Tolerance for mutations and chemical modifications in a siRNA”Nucl. Acids Res. 31:589-595; Holen et al. (2003) “Similar behavior ofsingle-strand and double-strand siRNAs suggests that they act through acommon RNAi pathway” Nucl. Acids Res. 31:2401-2407; and Schwarz et al.(2002) Mol. Cell. 10:537-548.

Due to currently unexplained differences in efficiency between siRNAscorresponding to different regions of a given target mRNA, severalsiRNAs are typically designed and tested against the target mRNA todetermine which siRNA is most effective. Interfering RNAs can also beproduced as small hairpin RNAs (shRNAs, also called short hairpin RNAs),which are processed in the cell into siRNA-like molecules that initiateRNAi (see, e.g., Siolas et al. (2005) “Synthetic shRNAs as potent RNAitriggers” Nature Biotechnology 23:227-231).

The presence of RNA, particularly double-stranded RNA, in a cell canresult in inhibition of expression of a gene comprising a sequenceidentical or nearly identical to that of the RNA through mechanismsother than RNAi. For example, double-stranded RNAs that are partiallycomplementary to a target mRNA can repress translation of the mRNAwithout affecting its stability. As another example, double-strandedRNAs can induce histone methylation and heterochromatin formation,leading to transcriptional silencing of a gene comprising a sequenceidentical or nearly identical to that of the RNA (see, e.g., Schramkeand Allshire (2003) “Hairpin RNAs and retrotransposon LTRs effect RNAiand chromatin-based gene silencing” Science 301:1069-1074; Kawasaki andTaira (2004) “Induction of DNA methylation and gene silencing by shortinterfering RNAs in human cells” Nature 431:211-217; and Morris et al.(2004) “Small interfering RNA-induced transcriptional gene silencing inhuman cells” Science 305:1289-1292).

Short RNAs called microRNAs (miRNAs) have been identified in a varietyof species. Typically, these endogenous RNAs are each transcribed as along RNA and then processed to a pre-miRNA of approximately 60-75nucleotides that forms an imperfect hairpin (stem-loop) structure. Thepre-miRNA is typically then cleaved, e.g., by Dicer, to form the maturemiRNA. Mature miRNAs are typically approximately 21-25 nucleotides inlength, but can vary, e.g., from about 14 to about 25 or morenucleotides. Some, though not all, miRNAs have been shown to inhibittranslation of mRNAs bearing partially complementary sequences. SuchmiRNAs contain one or more internal mismatches to the corresponding mRNAthat are predicted to result in a bulge in the center of the duplexformed by the binding of the miRNA antisense strand to the mRNA. ThemiRNA typically forms approximately 14-17 Watson-Crick base pairs withthe mRNA; additional wobble base pairs can also be formed. In addition,short synthetic double-stranded RNAs (e.g., similar to siRNAs)containing central mismatches to the corresponding mRNA have been shownto repress translation (but not initiate degradation) of the mRNA. See,for example, Zeng et al. (2003) “MicroRNAs and small interfering RNAscan inhibit mRNA expression by similar mechanisms” Proc. Natl. Acad.Sci. USA 100:9779-9784; Doench et al. (2003) “siRNAs can function asmiRNAs” Genes & Dev. 17:438-442; Bartel and Bartel (2003) “MicroRNAs: Atthe root of plant development?” Plant Physiology 132:709-717; Schwarzand Zamore (2002) “Why do miRNAs live in the miRNP?” Genes & Dev.16:1025-1031; Tang et al. (2003) “A biochemical framework for RNAsilencing in plants” Genes & Dev. 17:49-63; Meister et al. (2004)“Sequence-specific inhibition of microRNA- and siRNA-induced RNAsilencing” RNA 10:544-550; Nelson et al. (2003) “The microRNA world:Small is mighty” Trends Biochem. Sci. 28:534-540; Scacheri et al. (2004)“Short interfering RNAs can induce unexpected and divergent changes inthe levels of untargeted proteins in mammalian cells” Proc. Natl. Acad.Sci. USA 101:1892-1897; Sempere et al. (2004) “Expression profiling ofmammalian microRNAs uncovers a subset of brain-expressed microRNAs withpossible roles in murine and human neuronal differentiation” GenomeBiology 5:R13; Dykxhoorn et al. (2003) “Killing the messenger: ShortRNAs that silence gene expression” Nature Reviews Molec. and Cell Biol.4:457-467; McManus (2003) “MicroRNAs and cancer” Semin Cancer Biol.13:253-288; and Stark et al. (2003) “Identification of DrosophilamicroRNA targets” PLoS Biol. 1:E60.

The cellular machinery involved in translational repression of mRNAs bypartially complementary RNAs (e.g., certain mRAs) appears to partiallyoverlap that involved in RNAi, although, as noted, translation of themRNAs, not their stability, is affected and the mRNAs are typically notdegraded.

The location and/or size of the bulge(s) formed when the antisensestrand of the RNA binds the mRNA can affect the ability of the RNA torepress translation of the mRNA. Similarly, location and/or size of anybulges within the RNA itself can also affect efficiency of translationalrepression. See, e.g., the references above. Typically, translationalrepression is most effective when the antisense strand of the RNA iscomplementary to the 3′ untranslated region (3′ UTR) of the mRNA.Multiple repeats, e.g., tandem repeats, of the sequence complementary tothe antisense strand of the RNA can also provide more effectivetranslational repression; for example, some mRNAs that aretranslationally repressed by endogenous miRNAs contain 7-8 repeats ofthe miRNA binding sequence at their 3′ UTRs. It is worth noting thattranslational repression appears to be more dependent on concentrationof the RNA than RNA interference does; translational repression isthought to involve binding of a single mRNA by each repressing RNA,while RNAi is thought to involve cleavage of multiple copies of the mRNAby a single siRNA-RISC complex.

Guidance for design of a suitable RNA to repress translation of a giventarget mRNA can be found in the literature (e.g., the references aboveand Doench and Sharp (2004) “Specificity of microRNA target selection intranslational repression” Genes & Dev. 18:504-511; Rehmsmeier et al.(2004) “Fast and effective prediction of microRNA/target duplexes” RNA10:1507-1517; Robins et al. (2005) “Incorporating structure to predictmicroRNA targets” Proc Natl Acad Sci 102:4006-4009; and Mattick andMakunin (2005) “Small regulatory RNAs in mammals” Hum. Mol. Genet.14:R121-R132, among many others) and herein. However, due to differencesin efficiency of translational repression between RNAs of differentstructure (e.g., bulge size, sequence, and/or location) and RNAscorresponding to different regions of the target mRNA, several RNAs areoptionally designed and tested against the target mRNA to determinewhich is most effective at repressing translation of the target mRNA.

Further Details Regarding Polycystin Variants

Any of a variety of PC-1-L3 and/or PC-2-L1 polypeptides and/or codingPKD1L3 and/or PKD2L1 nucleic acids can be used in the present invention.These include human PC-1-L3 and/or PC-2-L1 taste receptor polypeptidesand/or coding PKD1L3 and PKD2L1 genes, murine PC-1-L3 and/or PC-2-L1taste receptor polypeptides and/or coding PKD1L3 and PKD2L1 genes, andhomologous polypeptides and coding nucleic acids from a domesticated orlivestock animal. Examples of such polypeptides and coding PKD2L1 genesare available, including PC-2-L1 and PKD2L1 for mice, humans and dogs.Examples of such sequences are provided in the Examples below and arefurther available in public databases. In addition, naturally occurringhomologues of these genes can readily be obtained simply by screeninggenomic or cDNA libraries for other organisms. These libraries arewidely available, and can also be made using standard techniques, e.g.,as taught in Sambrook or Ausubel.

The sequence of any PKD1L3 and/or PKD2L1 gene and coded polypeptide canalso be modified by standard methods to provide variants of suchavailable sequences, including conservative or non-conservativevariants. Any available mutagenesis procedure can be used to modify aPKD1L3 or PKD2L1 gene. Such mutagenesis procedures optionally includeselection of mutant nucleic acids and polypeptides for one or moreactivity of interest (e.g., increased responsiveness to tastant stimuli,which can be useful in producing transgenic animals, or for biosensorapplications). Procedures that can be used include, but are not limitedto: site-directed point mutatgenesis, random point mutagenesis, in vitroor in vivo homologous recombination (DNA shuffling), mutagenesis usinguracil containing templates, oligonucleotide-directed mutagenesis,phosphorothioate-modified DNA mutagenesis, mutagenesis using gappedduplex DNA, point mismatch repair, mutagenesis using repair-deficienthost strains, restriction-selection and restriction-purification,deletion mutagenesis, mutagenesis by total gene synthesis, double-strandbreak repair, and many others known to persons of skill. Mutagenesis,e.g., involving chimeric constructs, are also included in the presentinvention. In one embodiment, mutagenesis can be guided by knowninformation of the naturally occurring molecule or altered or mutatednaturally occurring molecule, e.g., sequence, sequence comparisons,physical properties, crystal structure or the like. In another class ofembodiments, modification is essentially random (e.g., as in classicalDNA shuffling).

Additional information regarding mutation is found in the followingpublications and references cited within: Arnold, Protein engineeringfor unusual environments, Current Opinion in Biotechnology 4:450-455(1993); Bass et al., Mutant Trp repressors with new DNA-bindingspecificities, Science 242:240-245 (1988); Botstein & Shortle,Strategies and applications of in vitro mutagenesis, Science229:1193-1201 (1985); Carter et al., Improved oligonucleotidesite-directed mutagenesis using M13 vectors, Nucl. Acids Res. 13:4431-4443 (1985); Carter, Site-directed mutagenesis, Biochem. J. 237:1-7(1986); Carter, Improved oligonucleotide-directed mutagenesis using M13vectors, Methods in Enzymol. 154: 382-403 (1987); Dale et al.,Oligonucleotide-directed random mutagenesis using the phosphorothioatemethod, Methods Mol. Biol. 57:369-374 (1996); Eghtedarzadeh & Henikoff,Use of oligonucleotides to generate large deletions, Nucl. Acids Res.14: 5115 (1986); Fritz et al., Oligonucleotide-directed construction ofmutations: a gapped duplex DNA procedure without enzymatic reactions invitro, Nucl. Acids Res. 16: 6987-6999 (1988); Grundstrom et al.,Oligonucleotide-directed mutagenesis by microscale ‘shot-gun’ genesynthesis, Nucl. Acids Res. 13: 3305-3316 (1985); Kunkel, The efficiencyof oligonucleotide directed mutagenesis, in Nucleic Acids & MolecularBiology (Eckstein, F. and Lilley, D. M. J. eds., Springer Verlag,Berlin)) (1987); Kunkel, Rapid and efficient site-specific mutagenesiswithout phenotypic selection, Proc. Natl. Acad. Sci. USA 82:488-492(1985); Kunkel et al., Rapid and efficient site-specific mutagenesiswithout phenotypic selection, Methods in Enzymol. 154, 367-382 (1987);Kramer et al., The gapped duplex DNA approach tooligonucleotide-directed mutation construction, Nucl. Acids Res. 12:9441-9456 (1984); Kramer & Fritz Oligonucleotide-directed constructionof mutations via gapped duplex DNA, Methods in Enzymol. 154:350-367(1987); Kramer et al., Point Mismatch Repair, Cell 38:879-887 (1984);Kramer et al., Improved enzymatic in vitro reactions in the gappedduplex DNA approach to oligonucleotide-directed construction ofmutations, Nucl. Acids Res. 16: 7207 (1988); Ling et al., Approaches toDNA mutagenesis: an overview, Anal Biochem. 254(2): 157-178 (1997);Lorimer and Pastan Nucleic Acids Res. 23, 3067-8 (1995); Mandecki,Oligonucleotide-directed double-strand break repair in plasmids ofEscherichia coli: a method for site-specific mutagenesis, Proc. Natl.Acad. Sci. USA, 83:7177-7181 (1986); Nakamaye & Eckstein, Inhibition ofrestriction endonuclease Nci I cleavage by phosphorothioate groups andits application to oligonucleotide-directed mutagenesis, Nucl. AcidsRes. 14: 9679-9698 (1986); Nambiar et al., Total synthesis and cloningof a gene coding for the ribonuclease S protein, Science 223: 1299-1301(1984); Sakamar and Khorana, Total synthesis and expression of a genefor the a-subunit of bovine rod outer segment guanine nucleotide-bindingprotein (transducin), Nucl. Acids Res. 14: 6361-6372 (1988); Sayers etal., Y-T Exonucleases in phosphorothioate-based oligonucleotide-directedmutagenesis, Nucl. Acids Res. 16:791-802 (1988); Sayers et al., Strandspecific cleavage of phosphorothioate-containing DNA by reaction withrestriction endonucleases in the presence of ethidium bromide, (1988)Nucl. Acids Res. 16: 803-814; Sieber, et al., Nature Biotechnology,19:456-460 (2001); Smith, In vitro mutagenesis, Ann. Rev. Genet.19:423-462 (1985); Methods in Enzymol. 100: 468-500 (1983); Methods inEnzymol. 154: 329-350 (1987); Stemmer, Nature 370, 389-91 (1994); Tayloret al., The use of phosphorothioate-modified DNA in restriction enzymereactions to prepare nicked DNA, Nucl. Acids Res. 13: 8749-8764 (1985);Taylor et al., The rapid generation of oligonucleotide-directedmutations at high frequency using phosphorothioate-modified DNA, Nucl.Acids Res. 13: 8765-8787 (1985); Wells et al., Importance ofhydrogen-bond formation in stabilizing the transition state ofsubtilisin, Phil. Trans. R. Soc. Lond. A 317: 415-423 (1986); Wells etal., Cassette mutagenesis: an efficient method for generation ofmultiple mutations at defined sites, Gene 34:315-323 (1985); Zoller &Smith, Oligonucleotide-directed mutagenesis using M13-derived vectors:an efficient and general procedure for the production of point mutationsin any DNA fragment, Nucleic Acids Res. 10:6487-6500 (1982); Zoller &Smith, Oligonucleotide-directed mutagenesis of DNA fragments cloned intoM13 vectors, Methods in Enzymol. 100:468-500 (1983); and Zoller & Smith,Oligonucleotide-directed mutagenesis: a simple method using twooligonucleotide primers and a single-stranded DNA template, Methods inEnzymol. 154:329-350 (1987). Additional details on many of the abovemethods can be found in Methods in Enzymology Volume 154, which alsodescribes useful controls for trouble-shooting problems with variousmutagenesis methods.

Kits

In an additional aspect, the present invention provides kits embodyingthe methods, composition, systems or apparatus herein. Kits of theinvention optionally comprise one or more of the following: (1) acomposition, system, system component as described herein; (2)instructions for practicing the methods described herein, and/or forusing the compositions or operating the system or system componentsherein; (3) one or more PC-1-L3 or PC-2-L1 polypeptide or complex orcoding nucleic acid; (4) a container for holding components orcompositions, and, (5) packaging materials.

EXAMPLES

The following Examples serve to illustrate, but not to limit theinvention. One of skill will recognize a variety of non-criticalparameters that can be changed to achieve essentially similar results.

Example 1 A Novel Ion Channel Preferentially Expressed in MammalianTaste Receptor Cells (PC-2-L1/PKD2L1)

Introduction

Taste transduction is one of the most sophisticated forms ofchemotransduction in animals (Avenet & Lindemann (1989) Perspectives intaste reception. 112, 1-8; Margolskee (1993) R. Bioessays 15, 645-650).Gustatory signaling is found throughout the animal kingdom, from simplemetazoans to the most complex of vertebrates; its main purpose is toprovide a reliable signaling response to non-volatile ligands. Mammalsare believed to have five basic types of taste modalities: salty, sour,sweet, umami (the taste of MSG), and bitter. Each of these is thought tobe mediated by distinct signaling pathways leading to receptor celldepolarization, generation of a receptor or action potential, andrelease of neurotransmitter and synaptic activity (Roper (1989) Ann.Rev. Neurosci. 12:329-353). Recently, the receptors for bitter, sweetand umami were cloned and shown to be encoded by two families ofG-protein coupled receptors (Nelson et al. (2001) “Mammalian sweet tastereceptors” Cell 106(3): 381-90; Nelson et al. (2002) “An amino-acidtaste receptor” Nature 416(6877): 199-202; Zhang et al. (2003) “Codingof sweet, bitter, and umami tastes: different receptor cells sharingsimilar signaling pathways” Cell; 112(3):293-301; Zhao et al. (2003)“The receptors for mammalian sweet and umami taste” Cell 115(3):255-66;Mueller et al. (2005) “The receptors and coding logic for bitter taste”Nature 434 (7030): 225-9. In contrast, most of the molecular componentsof sour or salty pathways remain unknown. Electrophysiological studiessuggest that sour and salty tastants modulate taste cell function bydirect entry of H+ and Na+ ions through specialized membrane channels onthe apical surface of the cell. Thus, ion channels selectively expressedin taste receptor cells are ideal candidates as mediators of salt andsour tastes. Alternatively, ion channels may function as the finalcritical signaling component in the activation of taste cells (alcin tothe role of TRPM5 in sweet, umami and bitter cells; Zhang et al. (2003)“Coding of sweet, bitter, and umami tastes: different receptor cellssharing similar signaling pathways” Cell 112(3):293-30).

The identification and isolation of taste signaling molecules, inparticular receptors, ion channels and signaling components, would allowfor pharmacological and genetic modulation of taste signaling pathways.For example, availability of receptor and channel molecules (which areaccessible from outside of the cell) would permit the screening for highaffinity agonists, antagonists, inverse agonists, and enhancers of tastecell activity. These could then be used in the pharmaceutical and foodindustry to custom tune, enhance, block, or modulate different tastes.In addition, these cDNAs serve as invaluable tools in the generation oftaste (tongue-brain) topographic maps of sensory coding, and thedissection of taste-induced behaviors. Here we report the cloning andcharacterization of a taste-specific ion channel.

Overview

To discover novel receptors, ion channels and other membrane signalingmolecules involved in signal transduction in taste receptor cells, wedeveloped a novel bioinformatics/molecular screening strategy. Ourapproach relied on two empirical assumptions: First, receptors and ionchannels are transmembrane proteins. Second, sensory receptors in thevisual, olfactory, touch and taste systems are often selectivelyexpressed in restricted numbers of tissues. Therefore, we searched themouse genome for transmembrane proteins, and then screened for thosewith restricted expression. Chosen molecules were subjected toexperimental validation by PCR amplification reactions using tastetissue and in situ hybridization studies against mouse tongues.

Using a Hidden Markov Model (TMHMM 2.0) and f_TMHMM (UCSD SupercomputingCenter, Bourne lab), we screened the entire Ensembl mouse genomedatabase for genes encoding putative transmembrane domains. In order todetermine the tissue distribution for the chosen candidate genes, weused mouse Expression Sequence Tag (EST) databases(www.ncbi.nlm.nih.gov/BLAST) to identify gene transcripts (i.e., cDNAs)expressed in 3 tissues/organs or less. PCR amplification primers werethen prepared against selected cDNAs and RT-PCR reactions using mRNAfrom taste and non-taste tissues were carried out. Candidatespreferentially expressed in taste receptor cells were used for RNA insitu hybridization against various taste papillae. This strategy led tothe isolation of a novel taste-specific ion channel.

Bioinformatics Screen

Using a Hidden Markov Model (TMHMM 2.0) and f_TMHMM (UCSD SupercomputingCenter, Bourne lab) we screened the entire Ensembl mouse genome databasefor genes encoding transmembrane domains. In order to determine thetissue distribution for candidate cDNAs encoding transmembrane proteins,we used mouse Expression Sequence Tag (EST) databases as an expressionfilter (www.ncbi.nlm.nih.gov/BLAST); each cDNA expressed in 3tissues/organs or less, was chosen for further study.

Summary of results: (1) We identified 13,742 predicted and annotatedtranscripts encoding candidate transmembrane domains (Ensembl versionmm.30). (2) 1077 genes were selected by EST analyses as being expressedin 3 tissues or less. (3) 884 genes were chosen and subjected to tasteversus non-taste RT-PCR reactions using primers against the last exonand/or the 3′ untranslated region (primers were designed usinghttp://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi). (4) 26candidates were chosen for detailed in situ analysis.

Tissue Collection & RT-PCR Screen:

In order to determine if candidate cDNAs were selectively expressed intaste receptor cells—a goal of this example—we performed RT-PCRreactions using mRNA from taste and non-taste tissue.

Peeled, hand-dissected circumvallate and foliate taste papillae from ˜20mice were collected for each mRNA extraction (total of ˜120 mice wereused). Tissue was stored in RNAlater (Qiagen), and mRNA was extractedusing Micro-FastTrack 2.0 mRNA extraction kit (Invitrogen). cDNA wassynthesized using SuperScript II first-strand cDNA synthesis kit(Invitrogen) using oligo(dT) as primers. cDNA synthesis and progress wasmonitored by using T1R3 (Nelson et al., 2001) and GAPDH as controls.

RT-PQR experiments were performed using a minimum of two independent RTpreparations, each containing a mix of circumvallate and folliate mRNA(taste mRNA). As counter-screen, we sampled each candidate cDNA in twoindependent RT reactions using tongue epithelium devoid of tastereceptor cells (non-taste mRNA). 98 of the 884 candidates showedspecific RT-PCR reaction products in taste samples but not in any of thenon-taste reactions.

Data-Mining & RNA In Situ Hybridization:

Candidates shown to be selectively enriched in taste tissue by RT-PCRwere examined in detail using BLAST (http://www.ncbi.nlm.nih.gov/BLAST/)and motif search engines, and subjected to RNA in situ hybridizationsexperiments (see methods section in Hoon et al. (1999) “Putativemammalian taste receptors: a class of taste-specific GPCRs with distincttopographic selectivity” Cell 96:541-51 for details on in situpreparations). Male and female mouse tongues containing different tastepapillae were used in all in situ studies. Clone ID 529-30/597-8 wasshown to be expressed in selective subsets of taste receptor cells. FIG.1 shows results from the RNA in situ hybridization in circumvallatetaste papillae. Note the expression in subsets of taste cells, but notin surrounding non-taste tissue.

Clone ID529-30/597-8:

This gene was (a) isolated as one of the candidates of thebioinformatics screen, (b) found to be enriched in taste papillae usingour RT-PCR screen, and (c) shown to be expressed in a selective subsetof taste receptor cells.

Analyses of mouse, rat, and human sequence databases showed that theclone defined by PCR primers “CloneID529-30/597-8” encodes PKD2L1, adistant member of the Polycystin Kidney Disease family of proteins(Nomura, et al. (1998) “Identification of PKDL, a novel polycystickidney disease 2-like gene whose murine homologue is deleted in micewith kidney and retinal defects” J. Biol. Chem. 273:25967-25973),referred to as the TRPP family (Lin and Corey (2005) “TRP channels inmechanosensation” Curr Opin Neurobiol. 15(3):350-7. Review). PKD2L1 ismost similar to PKD2. The human gene was first identified by Wu et al.(Wu et al. (1998) “Identification of PKD2L, a Human PKD2-Related Gene:Tissue-specific Expression and Mapping to Chromosome 10q25” Genomics54(3) 564-568), and the mouse ortholog was isolated in a search for newmembers of the PKD family (Basora et al. (2002) “Tissue and CellularLocalization of a Novel Polycystic Kidney Disease-Like Gene Product,Polycystin-L” J. Am. Soc. Nephrol 13:293-301). An alignment of sequencesfor human, rat, and mouse PKD2L1 is provided in FIG. 2A-2B. Included inthe alignment in FIG. 2A is the match to a PCR fragment isolated fromtaste receptor cells (corresponding to exons 2-5), and used as the probein the in situ studies shown in FIG. 1A. FIG. 2B shows an alignment ofmouse to rat and human, along with percent identity calculations betweenmouse and rat (˜86% identical) and mouse and human (˜80% identical).

Mouse PKD2L1 fragment isolated from taste tissue (exons 2-5) (SEQ ID NO:1): DNA            ACAGCCGAGAACAGGGAGCTTTATGTCAAGACCACCCTGAGGGAGCTTGTGGTATACATAGTGTTCCTCGTGGACGTCTGTCTGTTGACCTACGGAATGACAAGTTCTAGTGCCTATTACTACACCAAAGTGATGTGTGAGTTGTTCCTACACACCCCATCCGACTCTGGAGTCTCCTTCCAGACCATCAGCAGCATGTCAGACTTCTGGGATTTTGCTCAGGGCCCACTCCTGGAGAGTTTGTACTGGACAAAGTGGTACAACAACCAGAGCCTGGGGCGTGGCTCCCACTCCTTCATCTACTATGAGAACCTGCTCCTGGGAGCCCCAAGGTTGCGGCAGCTGCGCGTGCGCAATGACTCCTGTGTGGTTCATGAAGACTTCCGGGAGGACATTTTGAACTGTTATGATGTGTACTCGCCGGACAAAGAAGATCAGCTCCCCTTTGGACCTCTGAACGGCACAGCGTGGACATACCATTCCCAGAATGAGCTGGGTGGCTCCTCCCACTGGGGCAGGCTCACAAGCTACAGCGGGGGTGGCTACTACTTGGATCTTCCAGGATCCCGACAAGCCAGTGCAGAGGCCCTCCAAGGACTCCAGGAGGGACTG

Taste tissue may also express PKD2L1 splice variants and may be presentin PKD2L1 cDNA libraries.

Predicted Amino Acid sequence (SEQ ID NO: 2)          TAENRELYVKTTLRELVVYIVFLVDVCLLTYGMTSSSAYYYTKVMSELFLHTPSDSGVSFQTISSMSDFWDFAQGPLLDSLYWTKWYNNQSLGRGSHSFIYYENLLLGAPRLRQLRVRNDSCVVHEDFREDILNCYDVYSPDKEDQLPFGPLNGTAWTYHSQNELGGSSHWGRLTSYSGGGYYLDLPGSRQASAEALQGLQEGL mouse PKD2L1 predictedmRNA (full-length, SEQ ID NO: 3)         ATGAAAGTATGGAAAGCCCCAAGAATCAGGAGCTACAAACCCTGGGGAACAGAGCCTGGGACAATCCTGCCTACAGCGACCCTCCTTCCCCGAACAGGACGCTGAGGATCTGCACTGTCTCCAGTGTGGCTCTCCCTGAGACTCAACCCAAAAAGCCAGAAGTCAGATGCCAGGAGAAGACACAGAGAACCCTGGTGTCCAGCTGCTGTCTCCATATCTGTCGGAGCATCAGAGGACTGTGGGGGACAACGCTGACTGAGAACACAGCCGAGAACAGGGAGCTTTATGTCAAGACCACCCTAAGGGAGCTTGTGGTATACATAGTGTTCCTCGTGGACGTCTGTCTGTTGACCTACGGAATGACAAGTTCTAGTGCCTATTACTACACCAAAGTGATGTCTGAATTGTTTCTACACACCCCATCCGACTCTGGAGTCTCCTTCCAAACCATCAGCAGCATGTCAGACTTCTGGGATTTTGCTCAGGGCCCACTCCTGGACAGTTTGTACTGGACAAAGTGGTACAACAACCAGAGCCTGGGGCGTGGCTCCCACTCCTTCATCTACTATGAGAACCTGCTCCTGGGAGCCCCAAGGTTGCGGCACGTGCGCGTGCGCAATGACTCCTGTGTGGTTCATGAAGACTTCCGGGAGGACATTTTGAACTGTTATGATGTGTACTCGCCGGACAAAGAAGATCAGCTCCCCTTTGGACCTCAGAACGGCACAGCGTGGACATACCATTCCCAGAATGAGCTGGGTGGCTCCTCCCAGTGGGGCAGGCTCACAAGCTACAGCGGGGGTGGCTACTACTTGGATCTTCCAGGATCCCGACAAGCCAGTGCAGAGGCCCTCCAAGGACTCCAGGAGGGACTGTGGCTGGACAGGGGCACTCGGGTGGTCTTTATCGACTTCTCCGTCTACAATGCCAACATCAATCTTTTCTGTATTCTGAGACTGGTGGTAGAGTTTCCAGCCACAGGAGGGACCATCCCATCCTGGCAGATCCGCACAGTTAAGCTGATCCGCTATGTGAATAACTGGGACTTCTTCATTGTGGGCTGTGAAGTTGTCTTCTGTGTCTTCATCTTCTATTATGTGGTGGAGGAAATCCTGGAAATCCACCTGCATCGGCTTCGCTACCTCAGCAGCGTCTGGAACATTCTGGACCTGGTGGTCATCTTGCTCTCCATCGTGGCTGTGGGTTTCCACATATTCCGAACCCTGGAAGTGAACCGACTGATGGGAAAGCTTCTGCAACAGCCAGACACGTATGCAGACTTTGAGTTCCTGGCCTTCTGGCAGACTCAGGACAATAACATGAACGCGGTCAACCTTTTCTTTGCTTGGATCAAGATATTCAAGTATATCAGCTTCAACAAGACCATGACACAGCTCTCCTCCACCCTGGCTCGATGTGCCAAGGACATCCTGGGCTTCGCAGTCATGTTCTTCATTGTCTTCTTCGCTTACGCCCAGCTTGGTTACCTGCTTTTTGGGACCCAAGTGGAAAACTTTAGCACTTTCGTCAAGTGCATTTTCACTCAGTTCCGGATAATCCTTGGGGATTTTGACTACAATGCCATCGACAATGCCAACAGAATCCTGGGCCCTGTGTACTTTGTCACCTATGTCTTCTTCGTCTTCTTTCGTGCTCCTGAACATGTTCCTGGCCATCATCAACGACACATACTCCGAGGTCAAGGAGGAGCTGGCTGGCCAGAAGGATCAGTTGCAGCTTTCTGACTTCCTGAAACAGAGCTACAACAAGACCCTACTAAGGCTGCGCCTGAGGAAAGAGCGGGTTTCTGATGTGCAGAAGGTCCTGAAGGGTGGGGAACCAGAGATCCAGTTTGAAGATTTCACCAGCACCTTGAGGGAACTGGGGCACGAGGAGCACGAGATCACCGCTGCCTTCACCAGGTTTGATCAGGATGGGGACCACATACTGGATGAGGAGGAGCAGGAACAGATGCGGCAGGGACTGGAAGAGGAGAGGGTGACCCTCAATGCTGAGATTGAGAACCTAGGCCGGTCTGTTGGACACAGCCCCCCAGGCGAATTGGGCGCGGAGGCTGCCAGAGGACAAAGCTGGGTTTCTGGAGAAGAATTCGACATGCTCACAAGGAGAGTTCTGCAGCTGCAGTGTGTTCTGGAAGGAGTTGTGTCCCAGATTGATGCTGTAGGCTCAAAGCTGAAGATGCTGGAGAGGAAAGGGGAGCTGGCTCCCTCCCCAGGAATGGGGGAACCAGCTGTTTGGGAGAACCTGTATAATCCGTCCTAGT human PKD2L1 taste predicted mRNAsequence (full-length, SEQ ID NO: 4):         ATGAATGCTGTGGGAAGTCGTGAGGGGCAGGAGCTGCAAAAGCTGGGGAGTGGAGCCTGGGACAACCCCGCCTACAGTGGTCCCCCTTCCCCACACGGGACGCTGAGAGTCTGCACCATCTCCAGCACGGGGCCTCTCCAGCCCCAACCCAAGAAGCCTGAAGATGAACCCCAGGAGACGGCATACAGGACCCAGGTGTCCAGCTGCTGCCTCCATATCTGTCAAGGCATCAGAGGACTTTGGGGAACAACCCTGACTGAGAACACAGCTGAGAACCGGGAACTTTATATCAAGACCACCCTGAGGGAGCTGTTGGTATATATTGTGTTCCTGGTGGACATCTGTCTACTGACCTATGGAATGACAAGCTCCAGTGCTTATTACTACACCAAAGTGATGTGTGAGCTCTTCTTACATACTCCATCAGACACTGGAGTCTCCTTTCAGGCCATCAGCAGCATGGCGGACTTCTGGGATTTTGCCCAGGGCCCACTACTGGACAGTTTGTATTGGACCAAATGGTACAACAACCAGAGCCTGGGCCATGGCTCCCACTCCTTCATCTACTATGAGAACATGCTGCTGGGGGTTCCGAGGCTGCGGCAGCTAAAGGTCCGCAATGACTCCTGTGTGGTGCATGAAGACTTCCGGGAGGACATTCTGAGCTGCTATGATGTCTACTCTCCAGACAAAGAAGAACAACTCCCCTTTGGGCCCTTCAATGGCACAGCGTGGACATACCACTCGCAGGATGAGTTGGGGGGCTTCTCCCACTGGGGCAGGCTCACAAGCTACAGCGGAGGTGGCTACTACCTGGACCTTCCAGGATCCCGACAGGGTAGTGCAGAGGCTCTCCGGGCCCTTCAGGAGGGGCTGTGGCTGGACAGGGGCACTCGAGTGGTGTTCATCGACTTCTCAGTCTACAATGCCAATATCAATCTTTTCTGTGTCCTGAGGCTGGTGGTGGAGTTTCCAGCTACAGGAGGTGCCATCCCATCCTGGCAAATCCGCACAGTCAAGCTGATCCGCTATGTCAGCAACTGGGACTTCTTTATCGTTGGCTGTGAGGTCATCTTCTGCGTCTTCATCTTCTACTATGTGGTGGAAGAGATCCTGGAGCTCCACATTCACCGGCTTCGCTACCTCAGCAGCATCTGGAACATACTGGACCTGGTGGTCATCTTGCTCTCCATTGTGGCTGTGGGCTTCCACATATTCCGAACCCTCGAGGTGAATCGGCTCATGGGGAAGCTCCTGCAGCAGCCAAACACGTATGCAGACTTTGAGTTCCTCGCCTTCTGGCAGACACAGTACAACAACATGAATGCTGTCAACCTCTTCTTCGCCTGGATCAAGATATTCAAGTACATCAGCTTCAACAAAACCATGACCCAGCTCTCCTCCACGCTGGCCCGCTGTGCCAAGGACATCCTGGGCTTCGCCGTCATGTTCTTCATTGTTTTCTTCGCCTATGCCCAACTCGGCTACCTGCTTTTCGGGACCCAAGTGGAAAACTTTAGCACTTTCATCAAGTGCATTTTCACTCAGTTCCGGATAATCCTCGGGGACTTTGACTACAATGCTATCGACAATGCCAACCGCATCCTGGGCCCTGCCTACTTTGTCACCTATGTGTCTTCGTCTTCTTCGTGCTCCTGAACATGTTCCTGGCCATCATCAATGACACATATTCAGAGGTCAAGGAGGAGCTGGCTGGACAGAAGGATGAGCTGCAACTTTCTGACCTCCTGAAACAGGGCTACAACAAGACCCTACTAAGACTGCGTCTGAGGAAGGAGAGGGTTTCGGATGTGCAGAAGGTCCTGCAGGGTGGGGAGCAGGAGATCCAGTTTGAGGATTTCACCAACACCTTAAGGGAACTGGGACACGCAGAGCATGAAATCACTGAGCTCACGGCCACCTTCACCAAGTTTGACAGAGATGGGAATCGTATTCTGGATGAGAAGGAACAGGAAAAAATGCGACAGGACCTGGAGGAAGAGAGGGTGGCCCTCAACACTGAGATTGAGAAACTAGGCCGATCTATTGTGTAGCAGCCCACAAGGCAAATCGGGTCCAGAGGCTGCCAGAGCAGGAGGCTGGGTTTCAGGAGAAGAATTCTACATGCTCACAAGGAGAGTTCTGCAGCTGGAGACTGTCCTGGAAGGAGTAGTGTCCCAGATTGATGCTGTAGGCTCAAAGCTGAAAATGCTGGAGAGGAAGGGGTGGCTGGCTCCCTCCCCAGGCGTGAAGGAACAAGCTATTTGGAAGCACCCGCAGCCAGCCCCAGCTGTGACCCCAGACCCCTGGGGAGTCCAGGGTGGGCAGGAGAGTGAGGTTCCCTATAAAAGAGAAGAGGAAGCCTTAGAGGAGAGGAGACTCTCCCGTGGTGAGATTCCAACGTTGCAGA GGAGTTAA Ensemblpredicts an ortholog in the Dog genome: geneID<ENSCAFG00000009644> (SEQID NO: 5)      MNAVESPEGQELQKMGSGAWDNPAYSGPPSPRGTLKICTISSAMPPQPQIQKPEDGPQEKAYRTLVSSCCFQICRGIRGLWGTTLTENTAENRELYVKTTLRELLVYIVFLVDICLLTYGMTSSSAYYYTKVMSELFLHTPSDTGVSFQAISSMADFWDFAQGPLLDSLYWTKWYNNQSLGHGSHSFIYYENLLLGVPRLRQLRVRNDSCVVHEDFREDILSCYDVYSPDKEEQLPFGPLNGTAWTYHSQDELGGSSHWGRLTSYSGGGYYLDLPGSRQASAEALQDLQEGLWLDRGTRVVFIDFSVYNANINLFCVLRLVVEFPATGGAIPSWQIRTVKLIRYVSNWDFFIIGCEIIFCIFIVYYMVEEILELHIHRLHYLSSIWNILDLVVIMLSIVAVGFHIFRTLEVNRLMGKLLQQPNMYADFEFLAFWQTQYNNMNAVNLFFAWIKIFKYISFNKTMTQLSSTLARCAKDILGFAVMFFIVFFAYAQLGYLLFGTQVENFSTFIKCIFTQFRIILGDFDYNAIDNANRLGPAYFVTYVFFVFFVLLNMFLAIINDTYSEVKEELAGQKDELQLSDLLKQGYNKTLLRLRLRKERVSDVQKVLQGGEQEIQFEDFTNTLRELGHAEHEITELTAAFTRFDQDGNHILDKKEQEQMQQDLEEKRVVLNAEIENLGQSIVSSSPGESGPEATRADGWVSGEEFYTLTRRVLQLETVLEGVMSQVDAVGSKLEMLERKEQLASSPGMGDQGIWEHLQPTSPVTPDPWGVQGGQESEFPGGREGE ALEEMRLS

Additional References

Liu et al. (2002) “Modulation of the human polycystin-L channel byvoltage and divalent cations” FEBS Letters 525 (1-3) 71-76; Keller etal. (1994) “Kidney and Retinal Defects (Krd), a Transgene-InducedMutation with a Deletion of Mouse Chromosome 19 That Includes the Pax2Locus” Genomics 23: 309-320; Gilbertson, T. (1993) The physiology ofvertebrate taste reception 3, 532-539; Kinnamon and Margolskee (1996),Curr. Opin. Neurobiol. 4:506-513; Adler et al. (2000) “A novel family ofmammalian taste receptors” Cell 100:693-702; Chandrashekar et al. (2000)“T2Rs function as bitter taste receptors” Cell 100:703-711.

Example 2 A Novel Gene Preferentially Expressed in Mammalian TasteReceptor Cells (PKD1L3)

To discover novel receptors, ion channels and other membrane signalingmolecules involved in signal transduction in taste receptor cells, wedeveloped a novel bioinformatics/molecular screening strategy. Ourapproach relied on two empirical assumptions: First, receptors and ionchannels are transmembrane proteins. Second, sensory receptors in thevisual, olfactory, touch and taste systems are often selectivelyexpressed in restricted numbers of tissues. Therefore, we searched themouse genome for transmembrane proteins, and then screened for thosewith restricted expression. Chosen molecules were subjected toexperimental validation by PCR amplification reactions using tastetissue and in situ hybridization studies against mouse tongues.

Overview

Using a Hidden Markov Model (TMHMM 2.0) and f_TMHMM (UCSD SupercomputingCenter, Boume lab), we screened the entire Ensembl mouse genome databasefor genes encoding putative transmembrane domains. In order to determinethe tissue distribution for the chosen candidate genes, we used mouseExpression Sequence Tag (EST) databases (www.ncbi.nlm.nih.gov/BLAST) toidentify gene transcripts (i.e., cDNAs) expressed in 3 tissues/organs orless. PCR amplification primers were then prepared against selectedcDNAs and RT-PCR reactions using mRNA from taste and non-taste tissueswere carried out. Candidates preferentially expressed in taste receptorcells were used for RNA in situ hybridization against various tastepapillae. Full-length clones were then isolated from cDNA librariesprepared from taste tissue and testis (testis usually express mostsensory-specific genes). This strategy led to the isolation of a PKD2-L1(PKD2-like 1), a member of the Polycystic Kidney Disease (PKD) family ofproteins selectively expressed in taste tissue (See, Example 1).

Members of the PKD family of genes belong to one of two independentsubgroups: PKD1s and PKD2s. Since PKD2s are often found in associationwith PKD1s (generally as heteromeric receptors/channels), we searchedfor PKD1-related family members in taste tissue. Using RT-PCR and RNA insitu hybridizations against taste papillae, we isolated and identifiedPKD1-L3 as a novel PKD selectively expressed in subsets of tastereceptor cells.

Bioinformatics Screen:

Using homology and literature searches we screened the mouse and humangenome databases for members of the PKD1 family of proteins. We thenperformed RT-PCR reactions with primers specifically targeting predictedexon regions for PKD1, PKD1-L1 (Yuasa et al., 2002), PKD1-L2 (Li et al.,2003), and PKD1-L3 (Li et al., 2003) using mRNA from taste tissue. Twosets of primers specific for PKD1-L3 produced correct PCR products intaste tissue but not in control non-taste epithelia.

RT-PCRs

Peeled, hand-dissected circumvallate and foliate taste papillae from ˜20mice were collected for each mRNA extraction (total of ˜120 mice wereused). Tissue was stored in RNAlater (Qiagen), and mRNA was extractedusing Micro-FastTrack 2.0 mRNA extraction kit (Invitrogen). cDNA wassynthesized using SuperScript II first-strand cDNA synthesis kit(Invitrogen) using oligo(dT) as primers. cDNA synthesis and progress wasmonitored by using T1R3 (Nelson et al., 2001) and GAPDH as controls.

RT-PCR experiments were performed using a minimum of two independent RTpreparations, each containing a mix of circumvallate and folliate mRNA(taste mRNA).

RNA In Situ Hybridization:

Candidates shown to be selectively enriched in taste tissue by RT-PCRwere cloned into plasmid vectors and used to generate specific probesfor RNA in situ hybridizations experiments (see methods section in Hoonet al., 1999 for details on in situ preparations). Male and female mousetongues containing different taste papillae were used in all in situstudies. FIG. 3 demonstrates that PKD1-L3 (probes ID “ex28-32” and“ex25” derived from exons 28-32 and exon 25, respectively) selectivelylabels taste receptor cells. Note the expression in subsets of tastecells, but not in surrounding non-taste tissue. FIG. 4 shows analignment of mouse, rat, and human PKD1-L3 protein sequences, includingcomputer-predicted exons.

REFERENCES

-   Hoon M A, Adler B, Lindemeier J, Battey J F, Ryba N J, Zuker C S    (1999). Putative mammalian taste receptors: a class of    taste-specific GPCRs with distinct topographic selectivity. Cell 96,    541-51-   Nelson, G., Hoon, M A., Chandrashekar, J., Zhang, Y., Ryba, N J.,    and Zuker, C S. (2001). Mammalian sweet taste receptors. Cell. 2001    Aug. 10; 106(3): 381-90.-   Lin, S Y, and Corey, D P. (2005). TRP channels in mechanosensation.    Curr Opin Neurobiol. 2005 May 25 (Epub ahead of print) Nomura, H.,    Turco, A E., Pei, Y., Kalaydjieva, L., Schiavello, T., Weremowicz,    S., Ji, W., Morton, C., Meisler, M., Reeders, S T., and    Zhou, J. (1998) Identification of PKDL, a novel polycystic kidney    disease 2-like gene whose murine homologue is deleted in mice with    kidney and retinal defects. J. Biol. Chem. 273 (1998), pp.    25967-25973.-   Wu, G., Hayashi, T., Park, J H., Dixit, M., Reynolds, D M., Li, L.,    Maeda, Y., Cai, Y., Coca-Prados, M., and Somlo, S. (1998)    Identification of PKD2L, a Human PKD2-Related Gene: Tissue-specific    Expression and Mapping to Chromosome 10q25. Genomics. vol 54(3) Dec.    15, 1998 pg. 564-568.-   Liu, Y., L1, Q., Tan, M., Zhang, Y Y., Karpinski, E., Zhou, J., and    Chen, X Z. (2002). Modulation of the human polycystin-L channel by    voltage and divalent cations. FEBS Letters Vol 525, Issues 1-3, Aug.    14, 2002, pages 71-76.-   Basora, N., Nomura, H., Berger, UV., Stayner, C., Guo L., Shen, X.,    and Zhou, J. (2002) Tissue and Cellular Localization of a Novel    Polycystic Kidney Disease-Like Gene Product, Polycystin-L. J. Am.    Soc. Nephrol 13:293-301, 2002.-   Li A, Tian X, Sung SW, and Somlo S. (2003) Identification of two    novel polycystic kidney disease-1-like genes in human and mouse    genomes. Genomics. 2003 June; 81(6): 596-608. Erratum in: Genomics.    2003 October; 82(4): 498-500.-   Yuasa T, Venugopal B, Weremowicz S, Morton CC, Guo L, and    Zhou J. (2002) The sequence, expression, and chromosomal    localization of a novel polycystic kidney disease 1-like gene,    PKD1L1, in human. Genomics. 2002 March; 79(3):376-86.

Mouse PKD1-L3 gene fragments isolated from taste tissue cdna: PKD1L3Exon25 (SEQ ID NO: 6):TCCACAAGCAAATGAAGTCGCCTCCCCAACATCAGGAGGACAGAGAGAACTATGGGGCTGGCTGGGTCCCCCCTGACACAAACATCACAAAAGTAGACAGTATTTGGCATTATCAGAATCAGGAGTCGCTGGGAGGCTATCCCATCCAAGGGGAGCTAGCCACTTACTCAGGAGGAGGCTATGTTGTGAGGCTTGGAAGAAACCACAGGGCG PKD1-L3 Exons28-32 (SEQ ID NO: 7):GGAAAAGGAACCTCCTGGACACAAGCATCGTCCTCATTAGCTTCAGCATCCTGGGCCTCAGCATGCAGAGCCTCTCTCTACTTCACAAAAAGATGCAGCAGTACCACTGTGACCGGGACAGGTTCATCAGTTTCTACGAGGCACTGAGAGTGAACTCTGCAGTCACCCACCTCAGGGGCTTCCTGCTTCTCTTCGCAACTGTGCGGGTCTGGGACCTACTGCGACATCATGCCCAGTTACAGGTCATCAACAAGACACTGTCCAAAGCCTGGGACGAGGTGCTGGGCTTTATACTGATCATCGTGGTCCTGTTAAGCAGCTATGCCATGACTTTCAACCTGCTGTTTGGATGGAGCATCTCTGACTACCAGAGCTTCTTCAGATCTATAGTGACTGTTGTTGGCCTCTTGATGGGAACTTCAAAGCACAAGGAGGTTATTGCTCTATACCCAATCCTGGGCTCCCTTTTGGTTCTCAGTAGCATCATCTTGATGGGACTTGTGATCATTAATCTTTTTGTTTCTGCCATTCTCATTGCCTTTGGGAAAGAAAGGAAGGCCTGTGAGAAAGAAGCTACACTGACAGATATGTTACTACAAAAGCTCTCAAGTCTGTTAGGAATCCGCCTGCACCAGAATCCATCTGAGGAACA CGC PredictedAmino Acid sequences: PKD1L3 Exon 25 (SEQ ID NO: 8)HKQMKSPPQHQEDRENYGAGWVPPDTNITKVDSIWHYQNQESLGGYPIQG ELATYSGGGYVVRLGRNHRAPKD1L3 Exons 28-32 (SEQ ID NO: 9)KRNLLDTSIVLISFSILGLSMQSLSLLHKKMQQYHCDRDRFISFYEALRVNSAVTHLRGFLLLFATVRVWDLLRHHAQLQVINKTLSKAWDEVLGFILIIVVLLSSYAMTFNLLFGWSISDYQSEFRSIVTVVGLLMGTSKHKEVIALYPILGSLLVLSSIILMGLVIINLFVSAILIAFGKERKACEKEATLTDMLLQK LSSLLGIRLHQNPSEEHmouse PKD1L3 predicted mRNA (full-length) (SEQ ID NO: 10)ATGCTCTTGCAGAGGCGGTCCTGGCTCTGGGTGTACATTAGAATCGGTGTCATTCTGGGTGATATTTTGGGACGTAAACCAAGCATCCGGGAGCAACATGGGGGAAACAGCTGGTATCAGCTTAACAGACTTTTCTGTGACTTCCAGGAAGCAGATAACTACTGCCACGCCCAGAGAGGACGCCTAGCCCACACGTGGAACCCCAAGGTTCGGGGTTTCCTAAAAAGCTTCCTGAATGAAGAAACAGTGTGGTGGGTCAGGGGAAACCTGACGCTGCCCGGATCGCATCCAGGGATAAATCAGACAGGAGGTGATGACGTCTTAAGGAACCAAAAGCCTGGCGAGTGCCCTTCCGTGGTCACACACTCTAATGCTGTCTTCTCAAGATGGAACCTGTGCATAGAGAAGCATCATTTCATTTGCCAGGCTGCCGCCTTTCCCCCTCAAGGTGCAAGCATTTGGAGAAATGAGTTTGGTCCTGGTCCTCTGTTACCCATGAAAAGAAGAGGAGCTGAGACAGAGAGACATATGATCCCAGGAAATGGCCCCCCGTTAGCCATGTGTCACCAACCCGCTCCTCCTGAGCTTTTTGAGACATTGTGCTTTCCCATTGACCCAGCTTCTTCAGCACCTCCAAAAGCCACACACAGGATGACAATCACATCCCTAACTGGAAGGCCACAGGTGACATCAGACACACTTGCATCCAGCAGCCCACCACAGGGGACATCAGAGACACCTGCATCCAGCAGCCCACCACAGGTGACATCAGCCACATCTGCATCTAGCAGCCCACCACAGGGGACATCAGACACACCTGCATCCAGCAGCCCACCACAGGTGACATCAGCCACATCTGCATCTAGCAGCCCACCACAGGGGACATCAGACACACCTGCATCCAGCAGCCCACGACAGGTGACATCAGCCACATCTGCATCTAGCAGCCCACCACAGGGGACATCAGACACACCTGCATCCAGCAGCCCACCACAGGTGACATCAGCCACATCTGCATCTAGCAGCCCACCACAGGGGACATCAGACACACCTGCATCCAGCAGCCCACCAGAGGGGACATTAGACACACCTTCATCTAGCAGCCCACCACAGGGGACATCAGACACACCTGCATCCAGCAGCCCACCACAGGGGACATCAGAGACACCTGCATCCAACAGCCCACCACAGGGGACATCAGAGACACCTGGATTCAGCAGCCCACCACAGGTGACAACAGCCACACTTGTATCCAGCAGCCCACCACAGGTGACATCAGAGACACCTGCATCCAGCAGCCCAACACAGGTGACATCAGAGACACCTGCATCCAGCAGCGCAACACAGGTGACATCAGACACACGTGCATCCAATAGCCCACCACAGGGGACATCAGACACACCTGGATTCAGCAGCCCAACACAGGTGACAACAGCCACACTTGTATCCAGCAGCCCACCACAGGTGACATCAGACACACCTGCATCCAGCAGCCCACCACAGGTGACATCAGACACACCTGCATCCAGCAGCCCACCACAGGTGACATCAGAGACACCTGCATCCAGCAGCCCACCACAGGTGACATCAGACACATCTGCATCCATCAGCCCACCACAGGTAATATCAGACACACCTGCATCCAGCAGCCCACCACAGGTGACATCAGAGACACCTGCATCCAGCAGCCCAACAAACATGACATCAGACACACCTGCATCCAGCAGCCCAACAAACATGACATCAGACACACCTGCATCCAGCAGCCCAACAAACATGACATCAGACACACCTGCATCCAGCAGCCCACCATGGCCTGTTATAACAGAGGTCACCAGGCCTGAATCCACAATACCTGCTGGAAGATCTTTGGCAAACATCACTTCAAAGGCACAGGAAGACTCTCCCCTGGGAGTCATCTCTACCCATCCACAGATGTCATTTCAGAGTTCAACCAGTCAGGCCTTGGATGAGACAGCAGGGGAACGGGTCCCAACAATTCCTGATTTCCAAGCCCACAGTGAATTCCAGAAAGCTTGTGCCATCCTCCAGAGACTGAGAGACTTCCTGCCGACTTCTCCCACATCAGCTCAGGTCAGTGTGGCCAATTTACTCATTGACCTGAGTGAGCAGTTGCTGGTGCTCGCGTTTCAGAAGAACAACAGTTGGAGCTCTCAAACTCCAGCAGTCAGCTGCCCCTTCCAGCCTCTTGGACGTCTAACAACAACGGAAAAAAGCAGTCATCAGATGGCTCAGCAAGACATGGAACAGGTTGAAGACATGCTGGAGACATCCCTGATGGCCCTGGGGGAGATCCACAGAGCATTTTGCCAGGAGAGTCTGTGCCGTCAGTCAGCAGTGACCCTGGCCTCTCCCTCTGCTACTCTGATGTTGAGCAGCCAAAATGTGTCAACGTTGCCCCTGAGCACCTACACTTTGGGTGAGCCTGCACCCTTGACTTTGGGCTTCCCGTCAGCAGAAGCTCTGAAGGAGCTCTTGAACAAACACCCAGGGGTGAACCTTCAAGTGACAGGTCTGGCTTTCAACCCTTTTAAGACTTTGGATGACAAGAACATTGTTGGAAGCATTGGAAATGTGCAGCTGAGCTCTGCTTATCAGTCGATCAGAGTCCACGACTTAATAGAAGATATTGAGATCATGCTCTGGAGAAATGCCAGCATGGAGACCCAGCGCACCAGCCTCAACACAAGTACAGACCATTTCACAATCTCTGTGAACATCACTTCCTTGGAGAAGACCCTCATTGTGACCATCGAGCCTGAAAGTCCCCTCCTAATGACGCTCCACTTGGGCTTCCAGGACCAGCTGGCCCACACTCACTTCTATCTCAACATCAGCCTGCCAAGGGACCAAGTGTGGCAGAAAGATGAGGAGTACACGTGGGTGCTGACACCAGAGAACCTGTGGTACGGGACTGGCACCTACTACATAATGGCTGTGGAGAATAAAAGTACAGAGGCGGCACAGCACACACCCGTCCTGGTCTCAGTGGTCACAGCTGTCACCCAGTGCTATTTCTGGGACCGATACAATAGGACATGGAAGAGCGATGGATGCCAAGTGGGGCCGAAGAGCACCATTTTAAAGACACAGTGTCTCTGTGACCACCTGACCTTCTTCAGCAGCGACTTCTTCATCGTGCCGAGGACGGTGGATGTAGAAAACACCATCAAACTGCTTCTTCATGTGACCAACAACCCTGTCGGGGTGTCATTGCTGTCCAGCCTCCTAGGATTCTATATCCTCTTAGCCATGTGGGCTTCCAGAAAGGATCGAGAAGATATGCAGAAGGTGAAGGTAACAGTCCTGGCTGACAATGACCCCAGCTCTGCATCCCACTACCTTATCCAGGTCTACACTGGCTATCGGAGGAGGGCTGCTACCACCGCCAAGGTCGTTATCACTCTCTATGGCTCAGAGGGGCACAGTGAGCCCCAGCACCTTTGTGACCCTGAGAAGACAGTTTTTGAGCGTGGAGCACTGGATGTTTTCCTTCTTTCCACCGGATCCTGGCTGGGGGACCTGCATGGCCTTCGGCTGTGGCATGACAATTCTGGCGACAGCCCTTCTTGGTATGTAAGCCAGGTGATCGTCAGTGACATGACCACGAGGAAGAAATGGCATTTCCAGTGCAATTGTTGGCTGGCCGTGGACTTGGGCAACTGTGAGCGTGACAGGGTGTTCACACCAGCCTCCAGAAGCGAGCTCTCTTCCTTCAGACACCTGTTCTCCTCCACAATCGTAGAAAAGTTCACCCAGGATTATCTGTGGCTCTCAGTTGCAACTCGACATCCCTGGAACCAGTTTACACGAGTCCAGAGGCTCTCCTGCTGCATGGCACTACTGCTCTGTGACATGGTCATCAATATTATGTTCTGGAAGATGGGTGGCACCACTGCCAAGAGGGGCACCGAACAACTAGGTCCACTTGCTGTGACCTTGTCGGAGCTGCTCGTCAGCATCCAGACCTCCATCATCCTCTTCCCCATCCACCTCATCTTTGGGCGGCTCTTCCAGTTGATTCACCCACCAGAAGCTCTGCCCCAGCTTCCTTTCATCCAGGCTGCCTGGCCCCCTGCTCTTGTTTGTGAGTCCCCCTCTCTTACACAGGTGGTCAAGGAATTAAAGGAAACTGTCGGATTCCTGCTCAGGAGAAATACACAGCTGCTCTCGGAGTGTGAGCCGTCTTCGTGCAGTTCTTGTGACATTAACAAGCTGGCGAAGCTTTTATCCGGCCTCATTTACTGTCACTTAGAAGAGGAAGGCTGTCACCAGCAGACAGAATCCCACTGGGAAGACGCAGTGTCTGAAAACCATTACCATTTCTGCCGCTACCTTCTCCAACTTCTGCGGAGACTGAAAGCGCATTTAGAGGGTCTTGGTGCTACCCAGGATCACCAGTCTTGTGATTTCTCAGAAGCAGTCAGCCAACTTCAAAACCTCCAGGAACTCCTGGAGACACAGACTCTCCGCAGAGGGCCAGGGCCATGCAGGCATTCCACCAGTTTCCCCATCCTCAGCCCAGGAGAAGGGAAGAAGCCCATGTCATTTTGCCTGTTCAGATGGTTGAAGTGCAGCTGCTGGCTCCTTCTTGGTGTCATCAGCCTGGCCTCGGCCTTTTTTATAACGCTCTATAGCTTGGAGTTGGACAAAGACCAAGCCACCAGGTGGGTTATTTCAATGATGCTGTCGGTACTACAAGACATCTTTATCAGCCAGCCGATAAAGGTCATCTTCCTGACATTGTTGTTCTCCCTGATGGCAAACCACATGCCGTGGCTTAACAAAGACAAGGAACAACACGCCCGGAGAATCGTAGCACTTTGGGCAAAGTGTCCTTGGTCGGCACCTGGCTTGAGAGACAAGAACAATCCCATCTACACTGCCCCAGCAATGAACAACCTAGCCAAGCCTACAAGGAAGGCCTGGAAGAAGCAGCTCTCCAAGCTGACGGGTGGTACTCTGGTGCAAATCCTCTTCCTGACCCTGCTGATGACTACCGTCTATTCTGCAAAGGACTCTAGTCGATTTTTCCTCCATCGAGCTATCTGGAAGAGGTTTTCTCACCGTTTCTCAGAAATCAAAACTGTAGAGGATTTCTACCCCTGGGCCAACGGCACCCTCCTTCCTAACCTATATGGGGATTACAGAGGATTTATTACTGACGGGAACTCCTTTCTTCTGGGCAATGTTTTGATCCGCCAGACTCGCATTCCTAATGACATATTCTTCCCAGGATCTCTCCACAAGCAAATGAAGTCGCCTCCCCAACATCAGGAGGACAGAGAGAACTATGGGGCTGGCTGGGTCCCCCCTGACACAAACATCACAAAAGTAGACAGTATTTGGCATTATCAGAATCAGGAGTCGCTGGGAGGCTATCCCATCCAAGGGGAGCTAGCCACTTACTCAGGAGGAGGCTATGTTGTGAGGCTTGGAAGAAACCACAGTGCGGCAACCAGGGTTCTGCAGCATCTGGAACAGAGGCGCTGGCTGGACCACTGCACAAAAGCCCTCTTTGTAGAATTCACGGTCTTCAATGCTAATGTGAATCTGCTCTGTGCGGTGACCCTCATCTTGGAATCCAGTGGTGTGGGGACTTTCCTCACCTCCCTGCAACTGGACAGTTTAACTTCCCTTCAGTCATCAGAGAGGGGCTTCGCCTGGATCGTCTCACAGGTCGTCTACTACCTTCTCGTCTGTTACTATGCCTTCATCCAGGGCTGTCGGCTGAAGCGGCAGAGGCTGGCGTTCTTCACTAGGAAAAGGAACCTCCTGGACACAAGCATCGTCCTCATTAGCTTCAGCATCCTGGGCCTCAGCATGCAGAGCCTCTCTCTACTTCACAAAAAGATGCAGCAGTACCACTGTGACCGGGACAGGTTCATCAGTTTCTACGAGGCACTGAGAGTGAACTCTGCAGTCACCCACCTCAGGGGCTTCCTGCTTCTCTTCGCAACTGTGCGGGTCTGGGACCTACTGCGACATCATGCCCAGTTACAGGTCATCAACAAGACACTGTCCAAAGCCTGGGACGAGGTGCTGGGCTTTATACTGATCATCGTGGTCCTGTTAAGCAGCTATGCCATGACTTTCAACGTGCTGTTTGGATGGAGCATCTCTGACTACCAGAGCTTCTTCAGATCTATAGTGACTGTTGTTGGCCTCTTGATGGGAACTTCAAAGCACAAGGAGGTTATTGCTCTATACCCAATCCTGGGCTCCCTTTTGGTTCTCAGTAGCATCATCTTGATGGGACTTGTGATCATTAATCTTTTTGTTTCTGCCATTCTCATTGCCTTTGGGAAAGAAAGGAAGGCCTGTGAGAAAGAAGCTACACTGACAGATATGTTACTACAAAAGCTCTCAAGTCTGTTAGGAATCCGCCTGCACCAGAATCCATCTGAGGAACACGCTGACAACACTGGGTATTGA human PKD1L3 predicted mRNAsequence (full-length) (SEQ ID NO: 11):ATGTTCTTCAAAGGAGGAAGCTGGCTTTGGTTATACATCAGAACAAGTATTATTCTAGGAAGTGAGCTAAACAGCCCAGCACCACATGGGCAAAATAATTGTTACCAGCTTAACAGATTTCAATGCAGCTTTGAGGAAGCACAGCATTACTGTCATGTGCAGAGAGGATTCCTAGCTCATATTTGGAACAAGGAAGTTCAAGATCTCATCCGGGACTATCTGGAAGAAGGAAAGAAGTGGTGGATTGGGCAAAATGTAATGCCATTGAAAAAGCATCAAGACAACAAATACCCAGCAGACGTTGCAGCCAACGGGCCCCCAAAGCCCCTCAGCTGCACCTACCTGTCCAGAAACTTCATTCGGATCTCATCCAAAGGGGACAAGTGCTTACTGAAATACTATTTCATTTGCCAGACTGGTGACTTTTTGGACGGAGATGCCCATTATGAAAGAAATGGAAATAATTCCCATTTGTACCAGAGACACAAGAAGACAAAAAGAGGAGTTGCAATAGCAAGAGACAAAATGCCCCCAGGACCTGGTCATCTTCCAACCACATGTCACTATCCTCTTCCTGCTCATCTTTCCAAGACCCTGTGTCATCCCATCAGCCAGTTTCCTTCAGTACTATCAAGTATCACATCACAGGTAACATCAGCCGCATCTGAACCCAGCAGCCAGCCTCTCCCTGTGATAACACAGCTCACCATGCCCGTGTCTGTCACGCATGCTGGGCAATCTCTGGCAGAAACAACTTCAAGCCCAAAGGAAGAAGGTCATCCGAATACCTTCACCTCTTATCTACAAGTGTCATTGCAGAAGGCATCTGGTCAGGTCATAGATGAGATAGCAGGGAACTTCAGCAGAGCAGTTCATGGTTTGCAAGCTCTTAACAAACTACAGGAAGCTTGTGAGTTCCTCCAGAAACTAACAGCCTTAACCCCAAGATTTTCTAAGCCAGCTCAGGTTAATCTCATCAATTCCCTTATTTACCTGAGTGAGGAGTTACTCAGGATCCCATTTCAGAACAACAACAGTCTGGGCTTCAAAGTTCCTCCAACTGTCTGCCCCTTTCATTCCCTCAACAATGTCACCAAAGCTGGAGAAGGAAGTTGGCTGGAATCCAAGCGTCATACTGAGCCGGTAGAAGACATCCTGGAAATGTCCTTGGTGGAGTTTGGGAATATCGGGGAAGCATTTCTAGAGCAGAACCAGTCTCCCGAGTCTTCAGTGACTTTGACCTGTGCCAATGCTACTCTGCTGCTGAGCAGACAAAACATATCAACTTTACCGCTGAGCTCTTACACTCTGGGTCACCCAGCCCCTGTGAGGCTAGGCTTTCCGTCGGCTTTAGCTTTGAAGGAGCTCTTGAATAAACATCCAGGAGTTAATGTCCAAATAACAGGACTAGCTTTCAATCCCTTCAAGGATTTGGACAACAGAAACATTGTTGGAAGCATTGGAAGTGTGTTACTAAGCGCTAATCGTAAATTGCTCCAAGTCCATGATTTAATGGAGGACATTGAGATCATGCTCTGGAGAAATGTTAGCTTGGAAACCCATCCCACCAGCCTCAACATGAGCACACATCAGCTTACAATCACAGTGAACGTCACTTCCTTGGAGAAATCCTTGATAGTGAGCATAGATCCTGACAGTCCCCTTTTAATGACACTCTACCTGGGGTTCCAGTATCAGCCTAACTGCACTCACTTCCACCTGAACATCACCCTTCCAAAGGATAAGGTGTGGCAAAAAGATGAGGAGTACACGTGGGTGCTGAATCCAGAGCATCTGCAGCACGGGATTGGCACCTACTATATAACAGCTGTGCTGAGTGAGAGGCAGGAGGGTGCTCAGCAGACACCCAGCTTGGTCTCGGTCATCACCGCCGTCACTCAGTGTTACTACTGGGAGATCCACAAGCAGACATGGAGCAGCGCCGGATGCCAAGTTGGGCCACAGAGCACAATTCTGAGGACACAGTGTCTCTGTAACCACCTGACCTTCTTTGCCAGCGACTTCTTTGTCGTGCCCAGGACCGTGAATGTTGAAGACACGATCAAACTGTTCCTTCGCGTGACCAACAATCCTGTTGGGGTGTCACTGCTGGCCAGCCTTTTAGGATTTTATGTGATCACAGTTGTGTGGGCTCGGAAAAAGGATCAAGCAGATATGCAGAAGGTGAAGGTCACTGTCCTGGCTGATAATGACCCCAGCGCTCAATTTCACTACCTTATTCAGGTCTACACCGGATATCGAAGAAGCGCTGCTACAACAGCTAAGGTTGTCATCACCCTCTATGGATCAGAGGGACGGAGTGAGCCCCATCACCTCTGTGACCCCCAGAAGACAGTCTTTGAACGAGGGGGCCTGGATGTCTTCCTTCTCACCACTTGGACCTCTCTAGGGAACCTGCACAGCCTTCGGCTCTGGCATGACAATTCTGGCGTCAGTCCCTCCTGGTATGTCAGCCAGGTAATTGTCTGTGACATGGCAGTTAAGAGGAAGTGGCATTTCCTGTGCAATTGCTGGCTGGCTGTGGACCTCGGAGACTGTGAGCTTGACCGGGTCTTCATCCCAGTTTCAAAGAGAGAGCTCTTTTCCTTTAGACATCTGTTTTCCTCCATGATTGTGGAAAAGTTCACCCAGGATTATCTGTGGCTTTCAATTGCAACTCGGCATCCCTGGAACCAGTTTACAAGGGTCCAACGGCTGTCTTGCTGCATGACACTGCTACTCTGCAACATGGTCATCAATGTTATGTTCTGGAAGATAAACAGCACCACTGCCAAGAGAGATGAGCAAATGCGTCCATTTGCTGTGGCCTGGTCTGAACTGCTGGTCAGCATCCATACTGCTGTCATCCTCTTCCCAATCAATCTTGTCATAGGGCGGCTCTTCCCGTTGATTGAGCCACAGGAGACTCTGCCCCTCTTTCCTCCCATCCAGGCCTCCTGCCTCTCAGATGCTTCTGTTGAGCCTCTCTCTGCCACAATGGTAGTTGAGGAATTAAAGGAAACTGTGAGATTCCTGCTCAGGAGAAATACATACCTACTCTCCAAGTGTGAGCAGCCGCCATGGAGTTCTTGGGACATTACTAAGCTGGTGAAACTTTTATCCAGCCTCGTATCATCTCACTTGGAGGGTCAAGGCTGTCATCAGCAGGGAGAGCGCCACTGGGCACGTGTTGTTCCTGAAAACCACCATCATTTCTGCTGTTACCTGCATAGAGTTCTGCAGAGGCTGAAATCTCACTTAGGCACGCTGGGTCTCACCCAGGGTCACCAGTCCTGTGACTTCCTAGATGCAGCCAGCCAACTTCAAAAACTCCAGGAACTCTTGGAAACACATATTCTTCCCACGGAGCAAGAGCCATCCAGGGAAGTCACCAGTTTTGCCATCCTGAGCTCAGAAGAAGGAAAAAAGCCCATCTCAAATGGCCTGTCCAAATGGTTGACTTCAGTCTGCTGGCTCCTCTTAGGTTTCACTAGCCTGGCTTCAGCCTTTTTTACAGCACTTTATAGCTTGGAATTGAGCAAAGACCAAGCCACCAGCTGGATGATTTCAATTATTTTATCAGTGCTTCAGAACATCTTCATCAGCCAGCCAGTAAAGGTGGTCTTCTTCACATTCTTATACTCACTGATGATGAGCAGGATGCCACGGCTTAACAAAGAGAATGAACAACAAAGGATCTTGGCACTCTTGGCAAAATGTTCTTCGTCAGTACCAGGTTCAAGAGATAAGAACAACCCCGTCTATGTAGCCCCAGCTATAAATAGTCCAACTAAGCACCCAGAAAGAACCTTGAAAAAGAAGAAACTCTTCAAGCTGACTGGAGATATTTTGGTACAAATCCTCTTCCTTACCCTGTTGATGACTGCAATCTACTCTGCAAAGAACTCCAATAGATTTTACCTCCACCAAGCTATCTGGAAGACATTTTCGCACCAGTTCTCGGAAATCAAACTTCTTCAGGATTTCTACCCCTGGGCCAATCATATCCTTCTTCCTAGCCTGTATGGGGATTACAGAGGTAAGAATGCAGTCCTGGAGCCCAGTCATTGCAAATGTGGGGTACAATTAATTTTCCAAATACCCCGTACCAAGACCTATGAGAAAGTGGACGAAGGTCAGCTGGCGTTTTGTGATAACGGCCATACCTGTGGGCGTCCCAAGAGCCTATTCCCTGGACTTCATCTAAGGAGGTTCAGTTACATCTGTTCACCCAGGCCCATGGTGCTGATTCGCACTGATGAGCTTGACGAAAGGCTGACAAGCAAGAATGAGAATGGATTCAGTTACATCATGAGAGGTGCTTTCTTCACCTCTTTGAGACTGGAAAGCTTCACTTCCCTTCAGATGTCAAAGAAGGGCTGTGTCTGGTCTATCATCTCACAAGTCATCTATTATCTACTGGTCTGTTACTATGCCTTCATACAGGGTTGTCAGCTGAAACAGCAGAAGTGGAGGTTCTTCACTGGGAAAAGAAACATTCTGGACACAAGTATAATCCTCATTAGCTTCATCCTCCTGGGGCTTGACATGAAGAGTATTTCTCTACATAAGAAAAACATGGCACGATACCGCGATGACCAGGACAGATTCATCAGCTTCTATGAGGCAGTAAAAGTGAACTCTGCTGCGACTCACCTTGTGGGCTTCCCGGTTCTCCTGGCAACTGTTCAGTTATGGAACCTGCTGCGTCATAGCCCCAGGCTGCGGGTGATCAGCAGGACACTGAGCCGAGCCTGGGACGAGGTGGTGGGCTTTCTGCTGATCATCCTAATCCTGCTGACAGGCTATGCCATTGCCTTTAACCTGCTGTTTGGATGCAGCATCTCTGACTACCGGACATTTTTCAGCTCAGCAGTGACTGTTGTTGGTCTCCTGATGGGAATTTCTCACCAAGAGGAGGTTTTCGCTTTAGACCCAGTCCTGGGCACCTTTCTGATCCTCACCAGTGTCATCTTGATGGTACTTGTGGTAATTAATCTTTTCGTTTCGGCCATTCTCATGGCCTTTGGAAAAGAAAGAAAGTCGCTTAAGAAAGAAGCTGCACTAATAGATACACTGCTACAGAAGCTCTCAAATTTGTTAGGAATCAGTTGGCCCCAAAAAACCTCATCTGAGCAAGCAGCCACGACAGCAGTGGGCAGTGACACTGAAGTTTTAGATGAACTACCTTAA

Example 3 A Common Sensor for Acid Detection in the Tounge and SpinalCord

Mammals taste many compounds, yet use a sensory palette consisting ofonly five basic taste modalities: sweet, bitter, sour, salty, and umami(the taste of monosodium glutamate)^(1,2). While this repertoire mayappear modest, it provides animals with critical information about thenature and quality of food. Sour taste detection functions as animportant sensory input to warn against the ingestion of acidic (e.g.spoiled or unripe) food sources¹⁻³. We have used a combination ofbioinformatics, genetic, and functional studies to identify PKD2L1, apolycystic kidney disease-like ion channel⁴, as a candidate mammaliansour taste sensor. In the tongue, PKD2L1 is expressed in a subset oftaste receptor cells (TRCs) distinct from those responsible for sweet,bitter and umami taste. To examine the role of PKD2L1-expressing tastecells in vivo, we engineered mice with targeted genetic ablations ofselected populations of TRCs. Animals lacking PKD2L1-cells arecompletely devoid of taste responses to sour stimuli. Notably, responsesto all other tastants remained unaffected, proving that the segregationof taste qualities even extends to ionic stimuli. Our results nowestablish independent cellular substrates for four of the five basictaste modalities, and support a comprehensive labeled-line mode of tastecoding at the periphery⁵⁻¹⁰. Interestingly, PKD2L1 is also expressed inspecific neurons surrounding the central canal of the spinal cord. Herewe demonstrate that these PKD2L1-expressing neurons send projections tothe central canal, and selectively trigger action potentials in responseto decreases in extracellular pH. We show that these cells correspond tothe long sought components of the cerebrospinal fluid chemosensorysystem¹¹. Taken together, our results suggest a common basis for acidsensing in disparately different physiological settings.

A broad range of cell types, receptors and mechanisms have been proposedto mediate salt and acid sensing in TRCs¹⁻³. These include theactivation of ENaCs, ASICs, K2P channels, H⁺-gated calcium channels, aswell as the involvement of Na⁺—H⁺-exchangers, TRPV pain receptors, andeven acid-inactivation of K⁺-channels^(1-3,12-14). Significantly, mostof these proteins are broadly expressed in TRCs and other tissues. Incontrast, we previously isolated and characterized the receptors forsweet, umami and bitter taste^(5-7,15-17), and showed that each of thesethree taste modalities is mediated by highly selective receptor proteinsexpressed in distinct and independent populations of taste receptorcells⁵⁻¹⁰. Therefore, we reasoned that salt and sour taste should alsobe mediated by highly selective dedicated cells, and consequentlyexpected the receptor proteins to be very exclusive in their expressionpattern.

To identify novel taste receptors, we developed a multi-stepbioinformatics and expression screening strategy (see also, Examples 1and 2). First, since sensory receptors are expected to be membraneproteins, approximately 30,000 mouse open reading frames (ORFs) werescanned for the presence of at least one putative transmembrane segment.Second, because taste receptors are predicted to be very restricted intheir expression pattern, ORFs encoding candidate transmembrane proteinswere cross-searched against mouse EST databases to eliminate thosebroadly expressed. Next, to identify the subset specifically enriched intaste tissue, ORFs selected as encoding transcripts infrequentlyrepresented in EST databases (˜880 candidates) were used in RT-PCRreactions templated with mRNA from TRCs versus control tongueepithelium. Finally, given that our goal was to discover membraneproteins selectively expressed in subsets of TRCs (and ideally not insweet, bitter or umami sensing cells), we carried out detailed in situhybridizations against taste papillae. Of 26 cDNAs used in situ studies,five were found to robustly and selectively label subsets of TRCs. FIG.8 shows that one of these candidates, PKD2L1 is expressed in TRCs of alltaste papillae, including fungiform, circumvallate, foliate and palatetaste buds (further figure details are found below).

PKD2L1 encodes a polypeptide displaying significant amino acid sequencesimilarity to PKD2⁴, a gene mutated in many cases of autosomal dominantpolycystic kidney disease^(18,19). PKD2s are members of the TRPsuperfamily of ion channels²⁰, and have been recently shown to functionas non-selective cation channels when expressed in heterologouscells^(18,19,21). While the exact roles of PKDs remain unknown, they arebelieved to function as receptor/ion-channel complexes, often localizedto ciliated compartments, and implicated in sensing extracellularsignals (e.g. in renal epithelial cells^(18,19)). We reasoned that ifPKD2L1 has a specific role in taste it should be expressed insubpopulations of taste receptor cells with unique functionalcharacteristics. To determine which type of TRCs express PKD2L1, weperformed double labeling experiments with sweet, umami and bitter tastereceptors (T1R5 and T2R5), as well as TRPM5, the transduction channel ofsweet, bitter and umami sensing cells. Our results (FIG. 8) establishedthat PKD2L1 is expressed in cells distinct from those mediating sweet,umami and bitter taste (see also²²). FIGS. 1 and 8 show that PKD2L1 isexpressed in a novel population of TRCs. As shown in FIG. 8, in situhybridization (PKD2L1, PKD1L3, T1Rs, T2Rs and TRPM5) and double-labelfluorescent immunohistochemistry (PKD2L1) were used to directly examinethe overlap in cellular expression of taste receptors, TRPM5, PKD2L1 andPKD1L3. Panel A shows in situ hybridization of PKD2L1 and PKD1L3 againstcircumvallate, foliate, fungiform and palate taste buds, illustratingexpression of PKD2L1 in subsets of TRCs of all taste buds, but a totallack of PKD1L3 in fungiform and palate TRCs. Approximately 20% of tastecells express PKD2L1. Dotted lines show the outline of sample tastebuds. Panel (b) shows that PKD2L1 is not expressed in sweet, umami orbitter cells. The first three panels show co-labeling with a PKD2L1antisense RNA probe and T1R3 (T1R, sweet and umami cells), a mixture of20 T2Rs (bitter cells), and TRPM5 (sweet, umami and bitter cells),respectively. The last panel shows co-labeling with anti-PKD2L1antibodies and an antisense PKD1L3 RNA probe. Note the absence ofoverlap between PKD2L1-expressing cells and those expressing sweet,umami or bitter receptors. However, PKD1L3 is always co-expressed withPKD2L1 in CV and foliate papillae.

Mammalian taste receptor cells project specialized apical microvilli tothe taste pore, the site of interaction between tastants and tastereceptor proteins. All known taste receptor proteins localize to, andfunction, in this TRC compartment^(1,5-7,15,17,23). Therefore we wouldexpect bona-fide candidate receptors to also be enriched in the tastepore. We generated antibodies to PKD2L1 and used them inimmunofluorescence staining of tongue tissue sections. Examination ofCV, foliate and fungiform papillae demonstrated that PKD2L1 protein isindeed enriched in the apical surface of taste receptor cells, with theantibodies robustly labeling the taste pore region (FIG. 9). Theseresults implicate PKD2L1 as part of the taste sensing machinery.

PKD2 isoforms often require PKD1s for functional expression at the cellsurface^(18,19,21). The mammalian genome contains 4 members of the PKD1family: PKD1, PKD1L1, PKD1L2 and PKD1L3^(18,19). We performed in situhybridization studies with gene specific probes representing each familymember, and determined that PKD1L3 is specifically co-expressed withPKD2L1 in CV and foliate TRCs (FIG. 8, see also, Example 2, and²²). Wealso generated antibodies to PKD1L3 and demonstrated selectiveco-expression with PKD2L1 in non-TRPM5 expressing cells of the CV andfoliate (FIG. 9). Surprisingly, PKD1L3 transcript or protein is notdetectable in fungiform or palate taste buds (FIGS. 8 and 9), suggestingthat a different partner may be expressed in those TRCs.

If PKD2L1 is a mammalian taste receptor, we expect two basic criteria tobe met. First, PKD2L1-expressing TRCs should mediate a specific tastequality in vivo. Second, PKD2L1 protein should be activated in responseto taste stimuli.

To functionally dissect the role of PKD2L1-expressing cells in thetongue, we engineered mice where these cells were genetically ablated bytargeted expression of attenuated diphtheria toxin²⁴ (DTA). To validatethis approach as a means of uncovering TRC function, we first generatedmice where T1R2-regulatory sequences were used to target DTAexpressions. T1R2 is an essential subunit of the sweet receptorheterodimer (T1R2+3), and the selective ablation of these cells shouldgenerate animals with a specific loss of sweet taste^(6,9,10,17). Toinvestigate the taste responses of the genetically modified mice, werecorded tastant-induced action potentials from nerves innervating tastereceptor cells of the tongue; this physiological assay monitors theactivity of the taste system at the periphery, and provides an accurateand reliable measure of taste receptor cell function. Indeed, animalsexpressing DTA in T1R2 cells have an extraordinary loss of sweet, butimportantly retain umami, bitter, sour and salty tastes FIG. 5, panelA). These results further substantiate the exquisite segregation oftaste modalities at the periphery, and demonstrate the utility of usingDTA-mediated ablation of TRCs as a strategy for dissecting taste systemfunction. Next, we engineered animals where the PKD2L1 gene was used totarget Cre recombinase into PKD2L1-expressing cells; appropriateexpression was confirmed by performing double labeling with Cre andprobes specific to PKD2L1-cells, or by crossing them to GFP reporterlines²⁶. Mice expressing Cre in PKD2L1 cells were crossed to conditionalDTA lines, and double-positive progeny were scrutinized both for thespecificity and efficiency of killing, as well as the integrity of tastebuds. We checked the expression of T1Rs, T2Rs, and TRPM5^(8,27) incontrol and DTA-expressing animals, and found no significant differencesin the number or distribution of T1R- or T2R-positive cells between wildtype and ablated taste tissue. In contrast, the DTA-targeted mice had aprofound and practically complete loss of PKD2L1-expressing TRCs in thetongue. Remarkably, genetic ablation of the PKD2L1-expressing cellsproduces animals with a devastating loss of sour taste (FIG. 5, panels Aand B). Responses to all acid tastants, including citric acid, HCl,tartaric acid and acetic acid are completely abolished, with nosignificant activity over a range of 5 orders magnitude of protonconcentrations. However, responses to sweet, umami, bitter or saltytastants remain indistinguishable from wild type control animals. Theseresults firmly establish PKD2L1-expressing cells as the sour tastesensors, and further substantiate a model of coding at the periphery inwhich individual taste modalities operate independently of each other.

Acid sensing is important not only in the taste system, but also formonitoring the functional state of body fluids, including the internalmilieu of the brain. This is particularly well-studied in the centraland peripheral control of respiration, where pH sensing is the principalmechanism for monitoring CO₂ levels in the blood and cerebrospinalfluid^(11,28,29) (CSF). Thus, we wondered whether PKD2L1 might beexpressed in additional cell types, and if so whether such cells mayalso be involved in pH sensing in other physiological systems.

We carried out in situ hybridization and antibody staining experimentswith PKD2L1 on a wide range of other tissues and identified a singularadditional domain of expression: a discrete population of neuronssurrounding the central canal of the spinal cord, through its entirelength, from its origin in the brain stem to its end around the caudaequina (FIG. 6). Notably, these neurons send processes into the centralcanal, suggesting they may function as chemoreceptors sensing theinternal state of the CSF (FIG. 6, e.g., panels b and g¹¹). Given theiranatomical distribution and cellular morphology, we reasoned these cellsmight be part of the homeostatic circuitry responsible for monitoringand reporting the pH of the cerebrospinal fluid. This postulate predictsthat these neurons should trigger action potentials in response to acidstimulation. Therefore, we engineered mice where a GFP reporter wastargeted to PKD2L1-expressing cells, and performed patch clamprecordings from GFP-labeled cells in a spinal cord slice preparation³⁰.A priori, we anticipated some notable differences in the behavior ofthese cells compared to TRCs; while the taste system is tuned to respondto acid stimulation in the range of multiple pH units (i.e. pH 2-5), weexpected the CSF monitor cells to respond to pH changes within a rangeof a few tenths of deviation from pH 7.4. Indeed, FIG. 7 shows that thePKD2L1-expressing neurons display exquisite sensitivity and selectivityto pH stimulation. Exposure to test solutions between pH 6.5 and 7.4evoked a dramatic, dose dependent, and reversible increase in actionpotential (AP) frequency (FIG. 7). In contrast, the same acid stimulihave no significant impact on the response of control (e.g. unlabeled)cells, even after exposure to pH as low as 6.5 (lower pHs triggeredirreversible damage to the slice preparation).

Most of the known CSF-contacting neurons in mammals project ciliateddendrites into the CSP, where they are proposed to sense fluid flow,pressure, pH or the composition of the CSF¹¹. Our demonstration thatPKD2L1-expressing cells of the spinal cord selectively fire in responseto minor changes in proton concentration strongly suggests that theyfunction as sentinels of cerebrospinal and ventricular pH. Collectively,these results assign an entirely unexpected role to members of the PKDfamily of proteins, offer a new perspective into the potentialsignificance of PKD2s in health and disease, and bring forth asurprising unity in the cellular basis of pH sensing in very differentphysiological systems. It is useful to develop an activity assay forPKD2L1 to establish the molecular mechanism of acid activation, to studythe phenotype of PKD2L1 knockout animals, and determine whether PKD2L1functionally associates or interacts with different partners indifferent cells types. In this regard, it would be worth exploringwhether the differences in pH sensitivity between the tongue and spinalcord might be due to differences in PKD2L1-receptor complex composition.

The nature of the mammalian sour taste receptor and sour-sensing TRCshave been fertile ground for speculation over the years. A wide range ofcell types, receptors, and even receptor-independent mechanisms, havebeen proposed to mediate acid detection in the tongue¹⁻³. The resultspresented in this paper establish that sour taste, much like ourprevious findings for sweet, umami and bitter is mediated by a uniquecell type, independent of all other taste qualities. In addition, ourdemonstration that sour-less mice have normal salt responsesdemonstrates that salt taste is also mediated by independent TRCs.Together, these results impose a considerable revision of the currentviews of taste representation at the periphery, and make a compellingcase for a labeled line mode of coding across all five taste modalitiesand TRC types.

Accordingly, several lines of evidence now strongly implicate PKD2L1 asencoding a receptor protein. First, expressed PKD2L1 mRNA andpolycystin-2L1 selectively localize to the taste pore region of TRCs.Second, the presence of polycystin-2L1 protein functionally marks cellsas acid chemosensors, both in the tongue and in the nervous system.Finally, ablation of PKD211-expressing cells selectively eliminatespH-sensing in the tongue. It is of interest to further study thephenotype of PKD2L1 knockout animals, to establish the molecularmechanism of acid activation, and to further determine whether PKD2L1functionally associates or interacts with different partners indifferent cells types. In this regard, it would be worth exploringwhether the differences in pH sensitivity between the tongue and spinalcord might be due to differences in PKD2L1-receptor complex composition.

ADDITIONAL EXAMPLE DETAILS Molecular Cloning of PKD2L1

We used a strategy that combined bioinformatics and differentialscreening to isolate genes specifically expressed in taste receptorcells. Mouse genomic sequence information was obtained from EnsemblMm.30 (http://www.ensembl.org). Approximately 30,000 predicted proteinsequences were screened for the presence of at least one putativetransmembrane segment, using both TMHMM server version 2.0(http://www.cbs.dtu.dk/services/TMHMM-2.0/) and f_TMHMM (San DiegoSupercomputer center, http://www.sdsc.edu/pb/Group.html). The cDNAsequence for each candidate membrane protein was then extracted fromNCBI(http://www.ncbi.nlm.nih.govlblast/blastcgihelp.shtml#nucleotide_databases)and used to screen EST databases(http://www.ncbi.nlm.nih.gov/dbEST/index.html). Only EST hits withe-values of less than or equal to e⁻¹⁰⁰ were considered in our analysis.A total of 884 genes expressed in 3 tissues or less were chosen for PCRreactions with cDNA prepared from taste papillae mRNA (CV and foliate)and from surrounding non-taste epithelial tissue (non-taste control). Toensure specificity of the PCR reactions, all primers sets includedunique 3′UTR sequences(http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi.). A total of98 genes showed selective enrichment in taste versus non-taste tissue,and of these five were robustly expressed in subsets of TRCs. Fulllength clones were isolated from mouse taste cDNA libralies²³. See also,Examples 1 and 2.

In Situ Hybridization and Immunostaining

Fresh frozen sections (16 μm/section) were attached to silanized slidesand prepared for immunohistochemistry or in situ hybridization asdescribed previously²³. In situ hybridizations were carried out usingdigoxigenin or fluorescein labeled probes at high stringency(hybridization, 5×SSC, 50% formamide, 65-72° C.; washing, 0.2×SSC, 72°C.). For single-label detection, signals were developed using alkalinephosphatase-conjugated antibodies to digoxigenin and standardchromogenic substrates. Double-label fluorescent detection utilized analkaline phosphatase-conjugated anti-fluorescein antibody and ahorseradish peroxidase-conjugated anti-digoxigenin antibody incombination with fast red and tyramide fluorogenic substrates²³.

Anti-peptide antibodies to PKD2L1 (KLKMLERKGELAPSPGMGE), PKD1L3(DFQEADNYCHAQRGRLAHT), and TRPM5⁸ were generated in rabbits and purifiedas described previously³¹. Images were obtained using either a Leica SP2TSC or a Zeiss 510 Meta confocal microscope; 1-2 μm optical sectionswere recorded to ensure that any overlapping signal originated fromsingle cells. For double label experiments, in situ hybridization wascarried out before immunohistochemical detection.

Transgenic Animals

Transgenic lines were produced by pronuclear injection of zygotes fromFVB/N or CB6 (BALB/c×C57BL/6 hybrids) mice. The PKD2L1-IRES-Creconstruct was generated in RP23-297K23 and the T1R2—IRES-Cre inRP23-348G10 (http://bacpac.chori.org/) using a 4 kb IRES-Cre cassette(gift from Dr. Kevin Jones). Recombination was carried out exactly asdescribed previously³². All products were characterized by restrictionanalysis and direct sequencing to ensure fidelity of the recombinationevent and junctional sequences. Z/EG reporter lines²⁶ were obtained fromJackson Laboratories (Bar Harbor, Me.; Novak et al., 2000), andRosa26-flox-lacZ-flox-DTA animals²⁵ were a generous gift of Dr. DieterRiethmacher.

Nerve Recordings

Lingual stimulation and recording procedures were performed aspreviously described^(7,9). Neural signals were amplified (10,000×) witha Grass P511 AC amplifier (Astro-Med), digitized with a Digidata 1200BA/D converter (Axon Instruments), and integrated (r.m.s. voltage) with atime constant of 0.5 s. Taste stimuli were presented at a constant flowrate of 4 ml min⁻¹ for 20 s intervals interspersed by 2 min rinses withartificial saliva^(7,9) between presentations. All data analyses usedthe integrated response over a 25 s period immediately after theapplication of the stimulus. The mean response to 60 mM AceK was used tonormalize responses to each experimental series. Tastants used for nerverecordings were: 10 mM, 60 mM acesulfameK (AceK); 10 mM, 60 mM sodiumsaccharin (saccharin); 300 mM sucrose; 30 mM mono potassium glutamate+1mM inosine mono phosphate (Glu); 30 mM L alanine+1 mM inosine monophosphate (Ala); 10 mM quinine hydrochloride (Qui); 100 μM cycloheximide(Cyx); 10 mM 6-n-propyl 2-thiouracil (PROP); 50 mM, 100 mM sodiumchloride (NaCl); 10 mM, 50 mM citric acid; 10 mM, 50 mM tartaric acid;50 mM, 500 mM acetic acid; pH 2 hydrochloric acid (HCl); 10 mM citricacid pH 2, 4 and 6.

Spinal Cord Slice Recordings

Electrophysiological experiments were performed on P1-P4 mice aspreviously described³⁰. Spinal cord slices 250-300 μm thick weregenerated using a Vibratome® 3000 Plus at 0-4° C. in a modified Ringers'solution (0.5 mM CaCl₂, 3.7 mM MgSO₄). After at least a 1 h recoveryperiod, slices were transferred to a recording chamber and perfused withoxygenated Ringers' solution (pH 7.4) at room temperature. Loose-patchand whole-cell patch clamp recordings from GFP-labeled and unlabeledcells were performed using an EPC-10/2 amplifier and Patchmastersoftware (HBEKA Electronik). Slices were stimulated with a solutioncontaining 140 mM NaCl, 3 mM KCl, 1.3 mM MgSO₄, 2.5 mM CaCl₂, 10 mMglucose, 10 mM HEPES at various pH (7.4, 6.9, 6.5).

Additional Figure Details

FIG. 5A-B: PKD2L1-expressing TRCs are the mediators of sour taste. (a)Targeted expression of attenuated diphtheria toxin to selectivepopulations of TRCs produces animals with selective deficits in tasteresponses. Wild-type mice (WT) show robust neural responses to sweet(saccharin and acesulfameK, AceK), bitter (quinine), amino acid(glutamate), salty (NaCl) and sour tastants (citric acid, acetic acidand hydrochloric acid, HCl). However, ablation of sweet cells(T1R2-expressing TRCs) generates animals with a dramatic loss of sweettaste (middle panel). In contrast, ablation of PKD2L1-expressing cellseliminates responses to all acid stimuli (bottom panel). Importantly,responses to all other taste qualities remain unimpaired in theDTA-expressing animals. Shown are integrated chorda tympani responsesnormalized to the response to 60 mM AceK; see herein for details on theablated lines and concentrations of tastants. (b) Average neuralresponses of animals lacking PKD2L1-expressing cells to an expandedpanel of tastants; note normal responses to sweet, umami, bitter andsalt stimuli. Wild type, black bars; PKD2L1-DTA, outline bars. Thevalues are means±s.e.m. (n=5) of normalized chorda tympani responses.(c) Quantitation of acid responses of wild type (gray bars) andPKD2L1-DTA animals (outline bars). The values are means±s.e.m. (n=6).

FIG. 6: PKD2L1 is expressed in neurons contacting the central canal ofthe spinal cord. (a-b) Antibody labeling with anti-PKD2L1 antibodiesreveals a population of a population of PKD2L1 expressing neuronssurrounding the central canal of the spinal cord. (b) expanded view ofdotted area from panel (a). (c-d) The PKD2L1-expressing cells are foundthroughout the entire length of the spinal cord. Shown are in situhybridization experiments with PKD2L1 specific probes on a sagitalsection of a P1 mouse. Section shown corresponds approximately to boxedarea in panel (c). (e-f) PKD2L1-expression extends through the brainstem and into the IV ventricle (IV). There is also a very small group ofpositive cells in the hypothalamus. (g) PKD2L1-expressing neuronsproject into the central canal; note robust expression of PKD2L1receptors at the terminals. Shown are immunofluorescent stainings withanti-PKD2L1 antibodies; cc refers to central canal.

FIG. 7: PKD2L1-expressing neurons of the central canal fire actionpotentials in response to pH stimulation Spinal cord neurons werepatched using a loose patch configuration³⁰, tested for the presence ofbasal activity and recorded in the cell-attached configuration. (a)GFP-expressing (PKD2L1-positive cells) or unlabeled (control) cells wereexamined for pH responses under a perfusion regime consisting of pH 7.4,pH6.9, pH 7.4 and pH 6.5. (b) Shown are AP traces in a window of ˜10 secfollowing exposure to test solutions at pH 7.4, 6.9 and 6.5. Note thedramatic increases in firing frequency in GFP-labeled cells. (c) Datawas analyzed by examining records of ˜4 minutes at each pH condition;spike sorting software (Axon Instruments) was used to calculate APfrequencies. Basal activity ranged between 1-5 Hz. A minimum of 8GFP-labeled and 5 unlabelled cells were characterized for each stimuli.The values are means±s.e.m. normalized to basal activity at pH 7.4.

FIG. 8: PKD2L1 is expressed in a novel population of TRCs. In situhybridization (PKD2L1, PKD1L3, T1Rs, T2R5 and TRPM5) and double-labelfluorescent immunohistochemistry (PKD2L1) were used to directly examinethe overlap in cellular expression of taste receptors, TRPM5, PKD2L1 andPKD1L3. (a) In situ hybridization of PKD2L1 and PKD1L3 againstcircumvallate, foliate, fungiform and palate taste buds illustratingexpression of PKD2L1 in subsets of TRCs of all taste buds, but a totallack of PKD1L3 in fungiform and palate TRCs. Approximately 20% of tastecells express PKD2L1. Dotted lines show the outline of sample tastebuds. (b) PKD2L1 is not in sweet, umami and bitter cells. The firstthree panels show co-labeling with a PKD2L1 antisense RNA probe (PKD)and T1R3 (T1R, sweet and umami cells), a mixture of 20 T2Rs (bittercells), and TRPM5 (sweet, umami and bitter cells), respectively. Thelast panel shows co-labeling with anti-PKD2L1 antibodies and anantisense PKD1L3 RNA probe. Note the absence of overlap betweenPKD2L1-expressing cells and those expressing sweet, umami or bitterreceptors. However, PKD1L3 is always co-expressed with PKD2L1 in CV andfoliate papillae.

FIG. 9: PKD2L1 and PKD1L3 are enriched in the taste pore.Immunofluorescent stainings of mouse taste buds with PKD2L1 (left panel)and with PKD1L3 (right panel) antibodies. The pictures showsuperposition of fluorescent antibody signals on DIC images of tastetissue. Dotted lines illustrate the outline of a taste bud, and arrowspoint to the taste pore region

FIG. 10: Loss of selective TRCs in DTA-expressing animals. Upper diagramillustrates the strategy used to target DTA or GFP to selectivepopulations of TRCs. BAC constructs contained the entire T1R2 or PKD2L1genes with the IRES-Cre added downstream of the termination codon, butupstream of polyA-addition signals. In both cases, the transgenicconstructs included at least 50 Kb of flanking sequences upstream anddownstream of the target gene (see Methods). Fidelity of Cre andreporter expression in the correct cell types was confirmed by doublelabeling with a variety of TRC-specific gene probes. Lower panels showin situ hybridization experiments examining the presence of sweet(T1Rs), bitter (T2Rs) or PKD2L1-expressing cells in the two engineeredlines. Targeting of DTA to T1R2— or PKD2L1-expressing cells eliminatesover 95% of their respective TRC population. In situ hybridizationprobes were as in FIG. 8.

REFERENCES

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While the foregoing invention has been described in some detail forpurposes of clarity and understanding, it will be clear to one skilledin the art from a reading of this disclosure that various changes inform and detail can be made without departing from the true scope of theinvention. For example, all the techniques and apparatus described abovecan be used in various combinations. All publications, patents, patentapplications, and/or other documents cited in this application areincorporated by reference in their entirety for all purposes to the sameextent as if each individual publication, patent, patent application,and/or other document were individually and separately indicated to beincorporated by reference for all purposes.

1. A method of identifying a compound that binds to or modulates anactivity of a polycystin 1-like 3 (PC-1-L3) taste receptor polypeptide,or a polycystin-2-like 1(PC-2-L1)/PC-1-L3 polypeptide complex, themethod comprising: (a.) contacting a biological or biochemical samplecomprising the PC-1-L3 taste receptor polypeptide or PC-2-L1/PC-1-L3polypeptide complex with a test compound; and, (b.) detecting binding ofthe test compound to the PC-1-L3 taste receptor polypeptide orPC-2-L1/PC-1-L3 polypeptide complex, or modulation of the activity ofthe polypeptide or polypeptide complex by the test compound, therebyidentifying the compound that binds to or modulates the activity of thePC-1-L3 taste receptor polypeptide or PC-2-L1/PC-1-L3 polypeptidecomplex. 2-3. (canceled)
 4. The method of claim 1, wherein (a.)comprises contacting one or more biological sample comprising one ormore PC-1-L3 taste receptor polypeptide or PC-2-L1/PC-1-L3 polypeptidecomplex with a plurality of test compounds and wherein (b.) comprisesdetecting binding of the test compounds to the PC-1-L3 taste receptorpolypeptide or PC-2-L1/PC-1-L3 polypeptide complex, or modulation of theactivity of the polypeptide by the test compounds, thereby identifyingone or more compound that binds to or modulates the activity of thePC-1-L3 taste receptor polypeptide or PC-2-L1/PC-1-L3 polypeptidecomplex.
 5. The method of claim 1, wherein the polypeptide complex is ataste receptor polypeptide complex.
 6. The method of claim 1, whereinthe PC-1-L3) taste receptor polypeptide, or a polycystin-2-like1(PC-2-L1)/PC-1-L3 polypeptide complex is a component of an acidsensing, pH sensing, or sour tastant sensing receptor.
 7. (canceled) 8.The method of claim 1, wherein the test compound is selected from thegroup consisting of: naturally occurring compounds, ions, sour tastants,small organic molecules, peptides, peptide mimetics, an acid, a weakacid, CO, CO₂, acetic acid, a specific blocker of carbonic anhydrase,MK-417, an ion channel agonist, an ion channel antagonist, an ionchannel enhancer, a non-specific Ca⁺ channel blocker, Nifedipine andstructurally related compounds, Verapamil and structurally relatedcompounds, gadolinium and structurally related compounds, and astretch-induced channel blocker.
 9. The method of claim 1, wherein thetest compound enhances an activity of the PC-1-L3 taste receptorpolypeptide or PC-2-L1/PC-1-L3 polypeptide complex.
 10. The method ofclaim 1, wherein the test compound potentiates an activity of thePC-1-L3 taste receptor polypeptide or PC-2-L1/PC-1-L3 polypeptidecomplex.
 11. The method of claim 1, wherein the test compound inhibitsor blocks an activity of the PC-1-L3 taste receptor polypeptide orPC-2-L1/PC-1-L3 polypeptide complex.
 12. The method of claim 1, whereinthe PC-1-L3 taste receptor polypeptide is a human PC-1-L3 taste receptorpolypeptide, or wherein the PC-2-L1/PC-1-L3 complex is a humanPC-2-L1/PC-1-L3 polypeptide complex.
 13. The method of claim 1, whereinthe PC-1-L3 taste receptor polypeptide is a murine PC-1-L3 tastereceptor polypeptide or wherein the PC-2-L1/PC-1-L3 polypeptide complexis a murine PC-2-L1/PC-1-L3 complex.
 14. (canceled)
 15. The method ofclaim 1, wherein step (b.) includes detecting binding between thePC-1-L3 taste receptor polypeptide and a moiety selected from the groupconsisting of: a PC-2-L1 polypeptide, a potentiator of the PC-1-L3 tastereceptor polypeptide, an antagonist of the PC-1-L3 taste receptorpolypeptide, an agonist of the PC-1-L3 taste receptor polypeptide, aninverse agonist of the PC-1-L3 taste receptor polypeptide, a ligand thatspecifically binds to the PC-1-L3 taste receptor polypeptide, and anantibody that specifically binds to the PC-1-L3 taste receptorpolypeptide.
 16. The method of claim 1, wherein step (b.) includesdetecting binding between the PC-2-L1/PC-1-L3 polypeptide complex and amoiety selected from the group consisting of: a potentiator of thecomplex, an antagonist of the complex, an agonist of the complex, aninverse agonist of the complex, a ligand that specifically binds to thecomplex, and an antibody that specifically binds to the complex. 17-20.(canceled)
 21. The method of claim 1, wherein detecting binding of thetest compound to the PC-1-L3 taste receptor polypeptide or thePC-2-L1/PC-1-L3 polypeptide complex, or activity of the test compound onthe PC-1-L3 taste receptor polypeptide or the PC-2-L1/PC-1-L3polypeptide complex comprises detecting one or more of: detectingbinding between PC-2-L1 and PC-1-L3, formation or stability of thepolypeptide complex, H⁺ flux, Na⁺ flux, Ca²⁺ flux, ion flux, changes inan activity of an intracellular pH or ion sensor, depolarization of thecell, cell membrane voltage changes, cell membrane conductivity changes,a kinase activity triggered upon binding of a compound to the PC-1-L3taste receptor polypeptide, generation, breakdown or binding of aphorbol ester by the PC-1-L3 taste receptor polypeptide, binding ofdiacylglycerol or other lipids by the PC-1-L3 taste receptorpolypeptide, cAMP activity, cGMP activity, GTPgammaS binding,phospholipase C activity, activity of an enzyme involved in cellularionic balance, binding of PC-1-L3 to another PKD protein, or atranscriptional reporter activity.
 22. The method of claim 1, furthercomprising recombinantly expressing a PKD1-L3 gene in a recombinantcell, or both a PKD1-L3 gene and a PKD2L1 gene in a recombinant cell,wherein the biological sample is derived from the recombinant cell. 23.The method of claim 22, wherein the PKD1-L3 gene, or the PKD1-L3 and thePKD2L1 gene is or are heterologous to the recombinant cell. 24-25.(canceled)
 26. The method of claim 1, wherein the PC-1-L3 taste receptorpolypeptide or the PC-2-L1/PC-1-L3 complex is incorporated into abiosensor. 27-35. (canceled)
 36. A method of detecting a taste-inducedbehavior or physiological response modulated by a PC-1-L3 taste receptorpolypeptide or PC-2-L1/PC-1-L3 taste receptor polypeptide complex, themethod comprising: (a) introducing a heterologous PKD1-L3 taste receptorgene into an animal and expressing an encoded heterologous PC-1-L3 tastereceptor polypeptide in a taste bud of the animal; (b) providing aputative taste receptor tastant or modulator to the animal; and, (c)monitoring a feeding behavior or physiological response of the animal inresponse to the presence of the putative taste receptor tastant ormodulator. 37-52. (canceled)
 53. A method of modulating an activity of acell expressing a PC-2L1/PC-1L3 polypeptide complex, the methodcomprising contacting the cell with a compound that binds to ormodulates an activity of the complex. 54-56. (canceled)
 57. Arecombinant cell comprising a heterologous PKD1-L3 gene and aheterologous PKD2-L1 gene. 58-62. (canceled)
 63. An isolated orrecombinant polypeptide complex comprising at least one of: arecombinant PC-1-L3 polypeptide or a recombinant polycystin 2-L1polypeptide, the complex further comprising at least one of: a PC-1-L3polypeptide, a polycystin 2-L1 polypeptide, a recombinant PC-1-L3polypeptide or a recombinant polycystin 2-L1 polypeptide. 64-68.(canceled)
 69. A knock out non-human animal comprising a defect in anative PKD1-L3 taste receptor gene or a defect in native PKD1-L3 geneexpression. 70-81. (canceled)